Superfamily receptor chimeras, translocation assay for superfamily receptor ligands, and methods and kits for detecting and characterizing receptor ligands

ABSTRACT

The present invention relates to novel superfamily receptor chimeras, methods for using superfamily receptor chimeras, methods and kits for detecting cellular function and metabolic state, and the treatment of disease states involving defective receptor protein cytoplasm/nuclear translocation. In particular, this invention is preferably directed to chimeras of glucocorticoid receptor/superfamily receptor proteins.

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/325,178, filed Sep. 28, 2001, the contents of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of Invention

[0003] The present invention relates to novel superfamily receptor chimeras, methods for using superfamily receptor chimeras, methods and kits for detecting cellular function and metabolic state, and the treatment of disease states involving defective receptor protein cytoplasm/nuclear translocation. In particular, this invention is preferably directed to chimeras of glucocorticoid receptor/superfamily receptor proteins.

[0004] 2. Background

[0005] Steroid receptors mediate the action of a broad range of low molecular weight ligands, commonly referred to as hormones. These molecules influence a wide variety of human physiological processes, and also impact many human disease states, including cancer, heart disease, arteriosclerosis, arthritis, and inflammatory states. Low molecular weight hormones diffuse throughout the body and pass through cell membranes, either in a passive mode, or in some cases via active transport. Once in a cell, each hormone will interact with a cognate receptor; the receptor for each ligand recognizes and binds with a high affinity to its cognate hormone. This specific binding event induces conformational changes in the structure of the receptor, and activates the receptor for a variety of intracellular processes. The primary mechanism by which receptors act is by binding to specific regulatory sites in chromosomes, and regulating the rate of expression of target cellular genes. A secondary mechanism, referred to as “rapid” or “non-genomic” events, involves the interaction with specific cellular protein kinase signaling cascades.

[0006] The steroid, nuclear, and orphan receptor family now encompasses a superfamily of proteins, with approximately 70-75 distinct members in the mammalian species. Some members of this family do not have recognized cognate ligands, and thus are referred to as “orphan” receptors. A second group, characterized originally as “nuclear” receptors, are located almost exclusively in the nucleus, both in the presence and the absence of ligand. A third group display both cytoplasmic and nuclear distributions, although the precise partitioning may change with the addition of hormone.

[0007] The subcellular distribution of these receptors clearly plays a major role in their biological activity. Many receptors, such as the glucocorticoid receptor (“GR”), are in the cytoplasm in the absence of ligand, and thus do not interact with target genes. For those receptors that undergo a large-scale cytoplasmic to nuclear translocation, an assay of the compartmental distribution provides a potentially useful method to detect the presence of the cognate ligand in a given biological sample. There is a need for improved assays for ligand detection.

[0008] Many chimeric receptors have been described, but these fusions have been characterized and considered exclusively in terms of their gene activation potential (see, for example, Retinoid-X Receptor Signalling in the Developing Spinal Cord, Solomin, L., Johansson, C. B., Zetterstrom, R. H., Bissonnette, R. P., Heyman, R. A., Olson, L., Lendahl, U., Frisen, J., and Perlmann, T., Nature 395:398-402 (1998).).

[0009] Further, few steroid, nuclear, or orphan receptors manifest a distinct cytoplasmic to nuclear translocation in response to hormone stimulation. The vast majority of the molecules distribute to the nucleus, or in a mixed cytoplasmic/nuclear arrangement. Thus, use of a subcellular distribution assay to monitor hormone function has not heretofore been possible.

[0010] Applicants have solved these problems by the construction of a chimeric receptor, comprising the N-terminal domain of the glucocorticoid receptor and the C-terminal, or ligand binding, domain of a target superfamily receptor. The inventive receptor retains the cytoplasmic/nuclear translocation properties of the glucocorticoid receptor, but responds only to a ligand for the target receptor.

[0011] We describe here chimeric proteins that reside in the cytoplasm of untreated cells, but manifest complete translocation to the nucleus when cells are induced with a ligand for the ligand-binding domain of the chimeric receptor protein. The inventive chimeras form the basis for a new, in vivo translocation assay for superfamily receptor ligands, and has important implications for mechanisms of receptor subcellular trafficking.

[0012] Further, the inventive subject matter opens a completely new approach to the study of the function of superfamily receptors. In particular, when labeled with an appropriate fluorescent tag, such chimeric receptors permit the use of a real time subcellular translocation assay to monitor the presence and concentration of the cognate ligand in living cells in real time. The inventive cell-based assay provides a direct method to detect the presence and concentration of a given hormone or other ligand in a sample. Further, the inventive methods provide a less expensive and complicated approach to the process of screening for ligands for the orphan receptors and alternate ligands for the characterized receptors. The availability of chimeric proteins with the nuclear/cytoplasmic translocation properties of GR, and the ligand responsiveness of a superfamily receptor, provides a powerful translocation assay for known superfamily receptor ligands, for the identification of novel ligand analogues, and for the identification of ligands for orphan receptors.

SUMMARY OF THE INVENTION

[0013] The present invention relates to a method for making a recombinant nuclear translocation protein, comprising:

[0014] covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the cytoplasmic/nuclear translocation domain of the glucocorticoid receptor protein, (ii) a superfamily receptor DNA sequence coding for the ligand binding domain of a superfamily receptor protein, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera,

[0015] wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence; and

[0016] expressing said DNA chimera in an expression system to prepare said protein.

[0017] The present invention further relates to a protein produced by the process of:

[0018] covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the 540 end of the DNA sequence of the glucocorticoid receptor protein, through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence, (ii) a superfamily receptor DNA sequence coding for the 3′ end of the DNA sequence of a superfamily receptor protein, through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera,

[0019] wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence,

[0020] wherein said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence,

[0021] and wherein said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor are covalently connected by a DNA linker sequence; and

[0022] expressing said DNA chimera in an expression system to prepare said protein.

[0023] The present invention further relates to a nucleic acid chimera comprising:

[0024] a nucleic acid sequence which codes for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein; and

[0025] a nucleic acid sequence which codes for the ligand binding domain of a superfamily receptor protein.

[0026] The present invention further relates to a chimeric protein comprising two elements:

[0027] a glucocorticoid receptor 5′ end, encompassing the nuclear translocation domain and helix 1; and

[0028] a superfamily receptor 3′ end, encompassing the ligand binding domain and helix 3.

[0029] The present invention further relates to a method for detecting a ligand of a superfamily receptor protein, which comprises:

[0030] producing a nucleic acid vector encoding a nucleic acid chimera comprising three elements: a 5′ end of a glucocorticoid receptor, encompassing the nuclear translocation domain and helix 1, a 3′ end of a superfamily receptor, encompassing the ligand binding domain and helix 3, and a nucleic acid sequence for a marker protein domain;

[0031] transfecting a eukaryotic cell with said nucleic acid vector;

[0032] isolating a clonal population of cells that express a chimeric protein translated from said nucleic acid vector;

[0033] contacting said cells with a sample compound or composition; and

[0034] detecting the presence of cytoplasmic/nuclear translocation in response to a ligand of said ligand binding domain.

[0035] The present invention further relates to a method for determining the concentration of a ligand of a labeled chimeric superfamily receptor protein, which comprises:

[0036] producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain;

[0037] transfecting a eukaryotic cell with said nucleic acid vector;

[0038] isolating a clonal population of transfected cells that express a chimeric protein translated from said nucleic acid vector;

[0039] contacting said transfected cells with a sample;

[0040] scanning one or more test cell(s) to obtain signal data from said labeled protein;

[0041] converting said signal data to obtain the cellular location of said labeled protein in said test cell(s); and

[0042] analyzing said data using an analysis system having an algorithm to calculate changes in the distribution of said labeled protein between the cell cytoplasm and the cell nucleus of said test cell(s), said analysis system having the capability of providing an accurate reading of the concentration of a ligand.

[0043] Further, the present invention relates to a kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising:

[0044] a live-cell system which expresses a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; and

[0045] a detection system for the detection of the translocation of said marker protein.

[0046] The present invention further relates to a kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising:

[0047] a quantity of a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain;

[0048] a cell-free membrane system which restricts translocation of the chimeric protein when no ligand is bound to the ligand binding domain of said chimeric protein, and which permits translocation of the chimeric protein when the ligand binding domain of said chimeric protein is bound to its ligand; and

[0049] a detection system for the detection of the translocation of said marker protein.

[0050] The present invention further relates to a method for diagnosis of defects in the nuclear transportation process, which comprises:

[0051] producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain;

[0052] transfecting a set of suspected defective cells with said nucleic acid vector;

[0053] isolating a clonal population of said cells that express a chimeric protein translated from said nucleic acid vector;

[0054] contacting said cells with a ligand of said ligand binding domain; and

[0055] detecting the presence or absence of cytoplasmic/nuclear translocation in response to said ligand.

[0056] Finally, the present invention further relates to a method for treating defective translocation of a superfamily receptor protein from the cytoplasm to the nucleus of a cell, in an animal in need thereof, comprising:

[0057] producing a nucleic acid vector which is capable of being transcribed, and which encodes a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein and a nucleic acid sequence coding for the ligand binding domain of said superfamily receptor protein;

[0058] transfecting a target cell in said animal with said nucleic acid vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1 is a drawing which depicts the construction of a chimeric GFP-GR-RAR receptor.

[0060]FIG. 2 is a graph which depicts transcriptional transactivation by a chimeric GFP-GR-RAR receptor.

[0061]FIG. 3 is a series of photographs which depict subcellular trafficking of a chimeric GFP-GR-RAR receptor.

[0062]FIG. 4 is a photograph which depicts the interaction of a chimeric GFP-GR-RAR receptor with heat shock protein 90.

[0063]FIG. 5 is a drawing which depicts the construction of a chimeric GFP-GR-ER receptor.

[0064]FIG. 6 is a series of photographs which depict subcellular trafficking of wild type ER receptor.

[0065]FIG. 7 is a series of photographs which depict subcellular trafficking of a chimeric GFP-GR-ER receptor.

[0066]FIG. 8 is a chart which depicts sequence alignments of the DNA sequence surrounding helix 1 and helix 3 in several representative mammalian steroid receptor DNA sequences.

[0067]FIG. 9 is a chart which depicts sequence alignments of the DNA sequence surrounding helix 3 in 49 mammalian steroid receptor DNA sequences.

[0068]FIG. 10 is a drawing which depicts the relationship between the protein secondary structure domains in four chimeric molecules synthesized by Applicants.

DETAILED DESCRIPTION OF THE INVENTION

[0069] The inventive subject matter opens a completely new approach to the study of the function of superfamily receptors. In particular, when labeled with an appropriate fluorescent tag, such chimeric receptors permit the use of a real time subcellular translocation assay to monitor the presence and concentration of the cognate ligand in living cells in real time. The inventive cell-based assay provides a direct method to detect the presence and concentration of a given hormone or other ligand in a sample. Further, the inventive methods provide a less expensive and complicated approach to the process of screening for ligands for the orphan receptors and alternate ligands for the characterized receptors. The availability of chimeric proteins with the nuclear/cytoplasmic translocation properties of GR, and the ligand responsiveness of a superfamily receptor, provides a powerful translocation assay for known superfamily receptor ligands, for the identification of novel ligand analogues, and for the identification of ligands for orphan receptors.

Definitions

[0070] “Chimera” refers to a recombinant nucleic acid molecule generated by cloning portion(s) of one or more nucleic acid sequence(s) in-frame into one or more other nucleic acid sequence(s) to produce a single nucleic acid sequence capable of being transcribed into a polypeptide. The polypeptide produced by such a nucleic acid sequence chimera is referred to as a “chimeric protein” or “protein chimera”.

[0071] “Superfamily receptor” refers to the complete family of steroid, nuclear, and orphan receptor proteins having an identifiable ligand binding domain. The term is intended to encompass the known classic nuclear receptors, hormone receptors, and orphan receptors, as well as proteins having an identifiable ligand binding domain which may discovered in the future.

[0072] “GR” refers to the glucocorticoid receptor protein.

[0073] “ER” the estrogen receptor protein α.

[0074] “GFP” refers to green fluorescent protein.

[0075] “Marker protein domain” refers to a protein domain which is detectable based on inherent structural or functional characteristics, such as fluorescence. In this regard, a number of fluorescent proteins of various colors such as yellow, green, red, and blue are known, and are contemplated by this definition.

[0076] “LBD” refers to a ligand binding domain of a receptor protein.

[0077] “GR*” refers to a mutant GR having increased ligand binding affinity and which is fused to GFP.

[0078] “RAR” refers to the retinoic acid receptor.

[0079] “GR-RAR” refers to a chimera which encodes GR* and its associated GFP, wherein the LBD of wild-type GR is replaced by the LBD of RAR.

[0080] “ATRA” refers to all-trans retinoic acid.

[0081] “Dex” refers to dexamethasone.

[0082] “Hsp” refers to heat shock protein.

[0083] “Environmental sample” refers to a compound or composition to be screened for the presence or concentration of a ligand, and includes, without limitation, blood, serum, and tissue samples, as well as individual samples or libraries thereof.

[0084] A “cell-based” system is a system which is based upon the use of cells derived, isolated, or otherwise acquired from a living organism, and includes, for example, a blood or serum sample and a cell culture.

[0085] “Wild-type nucleic acid sequence” refers to the nucleic acid sequence(s) found in subjects having normal function in the protein transcribed from the nucleic acid sequence(s). It is to be understood that some variation in wild-type nucleic acid sequence is to be expected, and the salient feature is normal protein function. “Wild-type” nucleic acid sequence is to be distinguished from nucleic acid sequence(s) transcribing defective or loss-of-function proteins.

[0086] A nucleic acid sequence “capable of being transcribed” refers to a nucleic acid sequence which, when introduced into a target cell, forms a complete transcription complex. The resulting complete transcription complex may comprise the required elements as introduced with the exogenous nucleic acid sequence, as provided by the target cell, or a combination of introduced and existing elements.

[0087] “Isolated nucleic acid sequence” refers to a nucleic acid sequence which has been purified to at least about 95% homogeneity. This definition includes nucleic acid sequences that hybridize under stringent hybridization conditions with a gene, such as a cDNA molecule. This definition further includes a genomic sequence of interest comprising a nucleic acid sequence between the initiation codon and the stop codon, including all of the introns that are normally present in a native chromosome. It may further include the 3′ and 5′ untranslated regions found in a mature mRNA. It may further include specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 kb, but possibly more, of flanking genomic DNA at either the 5′ or 3′ end of the transcribed region. The genomic DNA may be isolated as a fragment of 100 kb or smaller; and substantially free of flanking chromosomal sequence. The genomic DNA flanking the coding region, either 3′ or 5′, or internal regulatory sequences as sometimes found in introns, contains sequences required for proper tissue and stage specific expression.

[0088] In the context of interactions with a chimeric protein of the present invention, “associate” refers to a stable covalent or non-covalent bond which exists between said chimeric protein in the absence of the ligand for the LBD of said receptor protein.

[0089] “Cell-free membrane system” refers to a membrane which is either a product of a living cell or tissue and has been isolated outside a living cell, or is not the product of a living cell or tissue.

[0090] “Effecting” refers to the process of producing an effect on biological activity, function, health, or condition of an organism in which such biological activity, function, health, or condition is maintained, enhanced, diminished, or treated in a manner which is consistent with the general health and well-being of the organism.

[0091] “Enhancing” the biological activity, function, health, or condition of an organism refers to the process of augmenting, fortifying, strengthening, or improving.

[0092] “Pharmaceutically acceptable salt, ester, or solvate” refers to a salt, ester, or solvate of a subject compound which possesses the desired pharmacological activity and which is neither biologically nor otherwise undesirable. A salt, ester, or solvate can be formed with inorganic acids such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, gluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthylate, 2-naphthalenesulfonate, nicotinate, oxalate, sulfate, thiocyanate, tosylate and undecanoate. Examples of base salts, esters, or solvates include ammonium salts; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; salts with organic bases, such as dicyclohexylamine salts; N-methyl-D-glucamine; and salts with amino acids, such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quarternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides, such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkyl halides, such as benzyl and phenethyl bromides; and others. Water or oil-soluble or dispersible products are thereby obtained.

Methods for Making Chimeric Receptors

[0093] Multicellular organisms require specific intercellular communication to organize the complex body plan properly during embryogenesis, and to maintain its properties and functions during the entire life span. Small, hydrophobic signaling molecules, such as steroid hormones, certain vitamins, and metabolic intermediates, enter or are generated within target cells and bind to cognate members of a large family of nuclear receptors. Nuclear receptors (NRs) are of major importance for metazoan intercellular signaling, as they bring together different intracellular and extracellular signals to initiate and regulate gene expression programs. They act as transcription factors that: (1) respond directly through physical association with a large variety of hormonal and other regulatory, as well as metabolic signals; (2) integrate diverse signaling pathways as they are themselves targets of posttranslational modifications; and (3) regulate the activities of other signaling cascades, commonly referred to as signal transduction cross-talk. The genetic programs that they modulate affect virtually all aspects of the life of a multicellular organism, covering such diverse aspects as, for example, embryogenesis, homeostasis and reproduction, or cell growth and death.

[0094] Thus, there is a great need for both a better understanding of the functioning of superfamily receptors, and especially for the ability to successfully manipulate superfamily receptors to produce chimeric proteins useful for diagnostic and treatment purposes. With these needs in mind, the present invention relates to a method for making a recombinant nuclear translocation protein, comprising:

[0095] covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the cytoplasmic/nuclear translocation domain of the glucocorticoid receptor protein, (ii) a superfamily receptor DNA sequence coding for the ligand binding domain of a superfamily receptor protein, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera,

[0096] wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence; and

[0097] expressing said DNA chimera in an expression system to prepare said protein.

[0098] In a preferred embodiment, said method further comprises covalently connecting said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor utilizing a DNA linker sequence.

[0099] In a more preferred embodiment, said DNA linker sequence is a fragment of a polylinker sequence.

[0100] In another preferred embodiment, said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence.

[0101] In another preferred embodiment, said method further comprises covalently connecting said glucocorticoid receptor DNA sequence and said nucleic acid sequence for a marker protein utilizing a DNA linker sequence.

[0102] In another preferred embodiment, said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence of said glucocorticoid receptor DNA sequence.

[0103] In another preferred embodiment, said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence.

[0104] In another preferred embodiment, said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including DNA bases corresponding to about amino acid residue 570of said glucocorticoid receptor protein.

[0105] In another preferred embodiment, said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence.

[0106] In another preferred embodiment, said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence, the complete helix 3 sequence, and at most a fragment of helix 1 of said superfamily receptor DNA sequence.

[0107] Applicants have modified the ligand binding domain (LBD) of GFP-GR in such a way that the unique cytoplasmic/nuclear translocation activity of GR is maintained, but the modified chimera responds to a superfamily receptor ligand.

[0108] In developing such a system, Applicants overcame several potential problems. First, insofar as they were then understood, the nuclear localization signals of GR were embedded within the LBD and the C-terminal region of the DNA-binding domain. Thus, complete replacement of the GR LBD might disrupt the regulated translocation signals we intended to utilize.

[0109] Second, GR interacts in the cytoplasm with a complex array of chaperon proteins, including Hsp90 and Hsp70, and ligand-dependent displacement of these proteins was thought to be intimately involved in the translocation process. Interaction domains critical for HSP90 binding are located within helix 1 of the GR LBD. Thus, removal or alteration of the GR LBD could have disrupted both the chaperone interaction domains and the nuclear localization signals in this region of the protein.

[0110] With this perspective, the inventive GFP-GR-RAR fusions were generated in which the GR LBD was replaced by the corresponding domain from RAR, preserving the GR helix 1 region intact. The chimeric molecules were then evaluated for transactivation potential and cellular distribution of the chimeric proteins. The chimeric protein behaved as an RA-dependent transactivator, but was nonresponsive to GR agonists.

[0111] The availability of crystal structures for the LBD of several receptors has led us to a compelling model for LBD structural rearrangements involved in ligand binding and coactivator interactions. In contrast, the receptor transitions responsible for intracellular trafficking have not been well understood in the art, and no general model has previously existed to account for the subcellular distribution of the steroid, nuclear, and orphan receptor superfamily.

[0112] The inventive chimeras provide a new method for elucidating mechanisms of ligand induced nuclear translocation. The ability to swap LBDs and obtain novel ligand-regulated translocation has important implications for the structural requirements necessary to maintain ligand-mediated translocation and will serve as a valuable tool in elucidating how the ligand-induced structural transition is transmitted from the LBD to domains of the chimeric protein critical for translocation.

[0113] Each member of the steroid, nuclear, and orphan receptor superfamily displays a distinct subcellular distribution, particularly in terms of the partitioning between the cytoplasmic and nuclear compartments. Some receptors, such as the glucocorticoid receptor, show primarily a cytoplasmic location in the absence of ligand, but translocate rapidly, and almost completely, to the nuclear compartment after hormone activation.

[0114] Nuclear hormone receptors form an evolutionarily related superfamily of proteins that are involved in many physiological processes and diseases. Because of this involvement, the nuclear hormone receptors are a superfamily of crucial medical significance. Many nuclear receptors bind hormones, mediating the transcriptional response to a ligand. Others, however, have no known ligand.

[0115] To date, about 70-75 different superfamily receptors, including both classical nuclear receptors with known ligands and so-called orphan receptors, which are receptors without a ligand or with an unknown ligand, have been identified. They constitute a family of transcription factors that share a modular structure of 5-6 conserved domains encoding specific functions. The most prominent distinction to other transcription factors is their capacity specifically to bind small hydrophobic molecules. These ligands constitute regulatory signals, which change the transcriptional activity of the corresponding superfamily receptor after binding. Superfamily receptors are classified by homology to other family members, with the DNA binding domain and the ligand-binding domain having the highest evolutionary conservation.

[0116] Genetic programs consist typically of several hundred genes that are expressed in a spatially and temporally controlled fashion. Superfamily receptors often act as master ‘switches’ to initiate specific genetic programs that, for example, lead to cell differentiation, proliferation or apoptosis, or regulate homeostasis. In the context of other programs these genetic activities support or initiate complex physiological phenomena, such as reproduction and organ function. Once activated by the cognate ligand, nuclear receptors regulate the primary and secondary target gene expressions that make up the corresponding physiological event. Throughout the life cycle of a multicellular organism, the coordinate interplay between programs defining cell fates in different tissues, organs and finally the entire body is at the foundation of the organism's development and subsistence.

[0117] Superfamily receptors are composed of 5-6 regions that have generally modular character. In general, the N-terminal A/B region harbors one or more autonomous transcriptional activation function (AF-1), which, when linked to a heterologous DNA binding domain, can activate transcription in a constitutive manner. When comparing superfamily receptors from different subfamilies and groups, the A/B region displays the weakest evolutionary conservation, and the distinction between the A and B regions is not always evident. A/B regions differ significantly in their length, ranging from 23 to about 550 amino acids.

[0118] The highly conserved domain C harbors the DNA-binding domain (DBD) of superfamily receptors that confers sequence-specific DNA recognition. The DNA-binding domain is mainly composed of two zinc-finger motifs. There are three prototypic DNA-binding modes of superfamily receptors: (1) a homodimer that binds to a palindromic response element, (2) an anisotropic heterodimeric complex on a DR1 direct repeat; and (3) a monomer that binds to an extended hexameric motif, the so-called NBRE.

[0119] The D region of superfamily receptors appears to correspond to a hinge between the highly structured C and E domains. It might allow the DNA and ligand-binding domains to adopt several different conformations without creating steric hindrance problems. Note in this respect that the C and the E regions contribute dimerization interfaces allowing some receptors to accommodate different heterodimerization partners and different types of response elements. Region D contains a nuclear localization signal (NLS), or at least some elements of a functional NLS. The intracellular localization of superfamily receptors is a result of a dynamic equilibrium between nuclear-cytoplasmic and cytoplasmic-nuclear shuttling. At equilibrium the large majority of receptors are nuclear, although the glucocorticoid receptor resides at cytoplasmic locations in the absence of its cognate ligand and translocates to the nucleus in a ligand-induced fashion.

[0120] A key feature of a superfamily receptor is its ligand-binding domain (LBD) in the E region. This domain is highly structured, and encodes a wealth of distinct functions, most of which operate in a ligand-dependent manner. The LBD often harbors a repression function, a major dimerization interface, and the ligand-dependent activation function AF-2. A structure-based sequence alignment of all known superfamily receptor primary amino-acid sequences strongly supports a common fold for all superfamily receptor LBDs, which has been fully confirmed when the crystal structures of multiple other NR LBDs were solved.

[0121] The general fold of nuclear receptors consists of a three-layered α-helical sandwich. Further structural features are one β-hairpin and connecting loops of variable lengths. The helices have been designated H1 to H12, starting with the most N-terminal H1, and form a hydrophobic cavity that accommodates the hydrophobic ligands. The LBD is structurally defined as the domain generated by the elements between the beginning of helix H1 and the end of helix H12. The E domain undergoes a significant conformational change upon ligand binding.

[0122] Some receptors possess at the C-terminus of the ligand binding domain a region F, which displays little evolutionary conservation. The role of the C-terminal region F is unknown. Recent literature suggests that the F region might play a role in coactivator recruitment to the E domain and in determining the specificity of the ligand binding domain coactivator interlace, perhaps by fine tuning the molecular events associated with the transcriptional properties of the E domain or the entire receptor.

[0123] As will be apparent to one of ordinary skill in the art, based on Applicants' teachings herein, some variation in the length and composition of the linker polypeptide regions is to be expected. Such minor variations, not significantly affecting the tertiary structure of the chimeric protein, are permitted in Applicants' methods of making the inventive DNA chimeras and corresponding proteins.

[0124] Further, as there are as many as six DNA codons for a single amino acid, it will also be apparent that variations in the DNA sequence which do not affect the protein sequence are inconsequential to the methods of the present invention. Such variations are to be expected in a population and are within the scope of the present inventive subject matter.

[0125] Finally, it will be apparent to one of ordinary skill in the art, based on Applicants' teachings herein, that variations in a protein sequence are also to be expected in a population, and are within the scope of the present inventive subject matter. For example, conservative substitutions are permissible in many regions affecting the general conformation, and not the binding specificity, of a protein.

Synthesis of Compounds of the Invention

[0126] The nucleic acid chimeras and chimeric proteins of the present invention may be readily prepared by standard techniques of molecular biology, utilizing techniques known to those of ordinary skill in the art, as described in greater detail herein.

[0127] The products and intermediates may be isolated or purified using one or more standard purification techniques known to one of ordinary skill in the art, including, for example, one or more of simple solvent evaporation, recrystallization, distillation, sublimation, filtration, polymerase chain reaction, Southern blotting, Northern blotting, Western blotting, chromatography, including thin-layer chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, FPLC, HPLC (e.g. reverse phase HPLC), column chromatography, flash chromatography, radial chromatography, trituration, salt precipitation, two-phase separation, polymer precipitation, heat denaturation, isoelectric separation, dialysis, and the like.

Proteins Produced by the Inventive Methods

[0128] An exemplary method for producing proteins according to the inventive method could encompass the following procedure: using conventional techniques well known in the art, a protein may be produced by the inventive methods by covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the 5′ end of the DNA sequence of the glucocorticoid receptor protein, through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence, (ii) a superfamily receptor DNA sequence coding for the 3′ end of the DNA sequence of a superfamily receptor protein, through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera,

[0129] wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence,

[0130] wherein said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence,

[0131] and wherein said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor are covalently connected by a DNA linker sequence; and

[0132] expressing said DNA chimera in an expression system to prepare said protein.

[0133] It should be recognized that one skilled in the art may vary the exemplary inventive methods as set forth herein. Such variations are expected to be within the scope of the inventive subject matter.

Nucleic Acid Chimeras

[0134] The present invention further relates to a nucleic acid chimera comprising:

[0135] a nucleic acid sequence which codes for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein; and

[0136] a nucleic acid sequence which codes for the ligand binding domain of a superfamily receptor protein.

[0137] In a preferred embodiment, said nucleic acid chimera additionally comprises a nucleic acid sequence for a marker protein domain.

[0138] In a more preferred embodiment, said marker protein domain encodes a fluorescent protein.

[0139] In another preferred embodiment, said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence of said glucocorticoid receptor DNA sequence.

[0140] In another preferred embodiment, said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence.

[0141] In another preferred embodiment, said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including DNA bases corresponding to about amino acid residue 570of said glucocorticoid receptor protein.

[0142] In another preferred embodiment, said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence.

[0143] In another preferred embodiment, said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence, the complete helix 3 sequence, and at most a fragment of helix 1 of said superfamily receptor DNA sequence.

[0144] In another preferred embodiment, said ligand binding domain is the ligand binding domain of estrogen receptor.

[0145] In a particularly preferred embodiment, said nucleic acid chimera is SEQ. ID NO. 1.

[0146] In another preferred embodiment, said ligand binding domain is the ligand binding domain of retinoic acid receptor.

[0147] In a particularly preferred embodiment, said nucleic acid chimera is SEQ. ID NO. 2.

Chimeric Proteins

[0148] The present invention further relates to chimeric protein comprising two elements:

[0149] a glucocorticoid receptor 5′ end, encompassing the nuclear translocation domain and helix 1; and

[0150] a superfamily receptor 3′ end, encompassing the ligand binding domain and helix 3.

[0151] In a preferred embodiment, said chimeric protein further comprises a marker protein domain.

[0152] In a particularly preferred embodiment, said marker protein domain encodes a fluorescent protein.

[0153] In another preferred embodiment, said ligand binding domain is the ligand binding domain of estrogen receptor.

[0154] In a particularly preferred embodiment, said chimeric protein is SEQ. ID NO. 3.

[0155] In another preferred embodiment, said ligand binding domain is the ligand binding domain of retinoic acid receptor.

[0156] In a particularly preferred embodiment, said chimeric protein is SEQ. ID NO. 4.

[0157] As discussed above, chimeric proteins of the present invention are produced using conventional techniques well known in the art.

Methods for Detecting a Ligand

[0158] The present invention further relates to a method for detecting a ligand of a superfamily receptor protein, which comprises:

[0159] producing a nucleic acid vector encoding a nucleic acid chimera comprising three elements: a 5′ end of a glucocorticoid receptor, encompassing the nuclear translocation domain and helix 1, a 3′ end of a superfamily receptor, encompassing the ligand binding domain and helix 3, and a nucleic acid sequence for a marker protein domain;

[0160] transfecting a eukaryotic cell with said nucleic acid vector;

[0161] isolating a clonal population of cells that express a chimeric protein translated from said nucleic acid vector;

[0162] contacting said cells with a sample compound or composition; and

[0163] detecting the presence of cytoplasmic/nuclear translocation in response to a ligand of said ligand binding domain.

[0164] In a preferred embodiment, said marker protein domain encodes a fluorescent protein.

Methods for Determining Concentration of a Ligand

[0165] The present invention further relates to a method for determining the concentration of a ligand of a labeled chimeric superfamily receptor protein, which comprises:

[0166] producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain;

[0167] transfecting a eukaryotic cell with said nucleic acid vector;

[0168] isolating a clonal population of transfected cells that express a chimeric protein translated from said nucleic acid vector;

[0169] contacting said transfected cells with a sample;

[0170] scanning one or more test cell(s) to obtain signal data from said labeled protein;

[0171] converting said signal data to obtain the cellular location of said labeled protein in said test cell(s); and

[0172] analyzing said data using an analysis system having an algorithm to calculate changes in the distribution of said labeled protein between the cell cytoplasm and the cell nucleus of said test cell(s), said analysis system having the capability of providing an accurate reading of the concentration of a ligand.

[0173] In a preferred embodiment, said marker protein domain encodes a fluorescent protein.

[0174] It will be apparent to one of ordinary skill in the art that there are a number of commercially available detection and analysis systems which may be interchangeably incorporated into the inventive methods. Such variations are contemplated to be within the scope of the inventive subject matter.

Kits for Screening an Environmental Sample

[0175] In addition, the present invention relates to a kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising:

[0176] a cell-based system which expresses a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; and

[0177] a detection system for the detection of the translocation of said marker protein.

[0178] In a preferred embodiment, said kit additionally comprises one or more compounds and/or compositions which stably associate with said chimeric protein in the absence of a ligand for the ligand binding domain of said chimeric protein, and which dissociates from said chimeric protein in the presence of a ligand for the ligand binding domain of said chimeric protein.

Cell-free Kits for Screening an Environmental Sample

[0179] In addition, the present invention relates to a kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising:

[0180] a quantity of a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain;

[0181] a cell-free membrane system which restricts translocation of the chimeric protein when no ligand is bound to the ligand binding domain of said chimeric protein, and which permits translocation of the chimeric protein when the ligand binding domain of said chimeric protein is bound to its ligand; and

[0182] a detection system for the detection of the translocation of said marker protein.

[0183] In a preferred embodiment, said kit additionally comprises one or more compounds and/or compositions which stably associate with said chimeric protein in the absence of a ligand for the ligand binding domain of said chimeric protein, and which dissociates from said chimeric protein in the presence of a ligand for the ligand binding domain of said chimeric protein.

Methods for Diagnosis of Nuclear Transport Defects

[0184] Translocation across the nuclear membrane involves a ligand-bound receptor, having at least one nuclear or cytoplasmic localization sequence, importin or exportin proteins, and the protein complex comprising the nuclear pore. For translocation to the nucleus from the cytoplasm, a ligand-bound receptor is recognized by an importin, which in turn is recognized by nuclear pore protein(s) and transported across the nuclear membrane. However, defects in this process are not readily distinguishable from other defects or impediments in the gene expression cascade, including, for example, improper ligand or receptor expression or folding; defective chaperoning processes; improper second messenger protein expression or folding; and defective transcription processes, to name but a few; all of these defects result in the same phenotypic defect: lack of gene expression. It would be very valuable to be able to identify, or exclude, nuclear transport defects from the long list of possible defects in the gene expression process.

[0185] The present invention thus relates to a method for diagnosis of defects in the nuclear transportation process, which comprises:

[0186] producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain;

[0187] transfecting a set of suspected defective cells with said nucleic acid vector;

[0188] isolating a clonal population of said cells that express a chimeric protein translated from said nucleic acid vector;

[0189] contacting said cells with a ligand of said ligand binding domain; and

[0190] detecting the presence or absence of cytoplasmic/nuclear translocation in response to said ligand.

Methods for Treating Translocation Defect Disorders

[0191] Finally, the present invention relates to a method for treating defective translocation of a superfamily receptor protein from the cytoplasm to the nucleus of a cell, in an animal in need thereof, comprising:

[0192] producing a nucleic acid vector which is capable of being transcribed, and which encodes a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein and a nucleic acid sequence coding for the ligand binding domain of said superfamily receptor protein;

[0193] transfecting a target cell in said animal with said nucleic acid vector.

[0194] A vector of the present invention capable of being transcribed may comprise an effective amount of a recombinant nucleic acid sequence or the protein product of such a recombinant nucleic acid sequence, in combination with a pharmaceutically acceptable carrier. The effective amount of a recombinant protein will generally comprise from about 0.1 mg to about 100 mg of the active agent per kilogram of patient body weight per day. The effective amount can vary depending upon the physical status of the patient and other factors well known in the art. Moreover, it will be understood that this dosage of active agent can be administered in a single or multiple dosage units to provide the desired therapeutic effect. If desired, other therapeutic agents can be employed in conjunction with those provided by the present invention.

[0195] The compositions of the invention are preferably delivered to the patient by means of a pharmaceutically acceptable carrier. Such carriers are well known in the art and generally will be in either solid or liquid form. Solid form pharmaceutical preparations which may be prepared according to the present invention include powders, tablets, dispersible granules, capsules, cachets and suppositories. In general, solid form preparations will comprise from about 5% to about 90% by weight of the active agent.

[0196] The compositions of the invention may be provided in any suitable dosage form known in the art. For example, the compositions may be incorporated into tablets, powders, granules, beads, chewable lozenges, capsules, liquids, or similar conventional dosage forms, using conventional equipment and techniques know in the art.

[0197] Further, the dosage form can be in the form of a bi-layer tablet composed of at least one extended-release layer and at least one immediate-release layer. Also, the bi-layer tablet can be coated for ease of administration or can be enteric coated to reduce any gastric irritation and the unpleasant “burping” produced by the vitamins and minerals. Also, multi-particulate design of extended-release and immediate-release components can be enteric coated and compressed into a tablet or filled into hard or soft gelatin capsules.

[0198] When preparing dosage forms incorporating the compositions of the invention, the compositions are normally blended with conventional excipients such as binders, including gelatin, pregelatinized starch, and the like; lubricants, such as hydrogenated vegetable oil, stearic acid, and the like; diluents, such as lactose, mannose, and sucrose; disintegrants, such as carboxymethyl cellulose and sodium starch glycolate; suspending agents, such as povidone, polyvinyl alcohol and the like; absorbent, such as silicon dioxide; preservatives, such as methylparaben, propylparaben, and sodium benzoate; surfactants, such as sodium lauryl sulfate, polysorbate 80, and the like; and colorants, such as F.D. & C dyes.

[0199] For preparing compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be used which are either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, and cachets. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents; it can also be encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active compounds. In the tablet the active compound is mixed with carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 5 or 10 to about 90 percent of the active ingredient. Suitable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, coca butter, and the like. The term “preparation” is intended to include the formulation of the active compounds with encapsulating material as carrier providing a capsule in which the active component (with or without other carriers) is surrounded by carrier, which is thus in association with it. Similarly, cachets are included. Tablets, powders, cachets, and capsules can be used a solid dosage forms suitable for oral administration. Liquid form preparations include solutions, suspensions, and emulsions. As an example, water or water/propylene glycol solutions for parenteral injection may be used. Liquid preparations can also be formulated in solution in aqueous polyethylene glycol solution. Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, i.e., natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0200] Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions. These particular solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit. Alternately, sufficient solid may be provided so that after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon, or other volumetric container. When multiple liquid doses are so prepared, it is preferred to maintain the unused portion of said liquid doses at low temperature, i.e., under refrigeration, in order to retard possible decomposition.

[0201] The solid and liquid forms may contain, in addition to the active material, flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The liquid utilized for preparing the liquid form preparation may be water, isotonic water, ethanol, glycerine, propylene glycol, and the like as well as mixtures thereof.

[0202] Preferably, the preparations are in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active components. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself or it can be the appropriate number of any of these in packaged form.

[0203] The quantity of active compound in a unit dose of preparation may be varied according to the particular application and the potency of the active ingredients. Determination of the proper dosage for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day if desired or at one time, morning, afternoon, night as well as biphasic, triphasic, etc. Controlled and uncontrolled release formulations are also contemplated.

[0204] Although the products of the invention are preferably intended for administration to humans, it will be understood that the formulation may also be utilized in veterinary therapies for other animals.

[0205] For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides or cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby to solidify.

[0206] The pharmaceutical preparations of the invention may include one or more preservatives well known in the art, such as benzoic acid, sorbic acid, methylparaben, propylparaben and ethylenediaminetetraacetic acid (EDTA). Preservatives are generally present in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition.

[0207] Useful buffers for purposes of the invention include citric acid-sodium citrate, phosphoric acid-sodium phosphate, and acetic acid-sodium acetate in amounts up to about 1% and preferably from about 0.05 to about 0.5% by weight of the pharmaceutical composition. Useful suspending agents or thickeners include cellulosics like methylcellulose, carageenans like alginic acid and its derivatives, xanthan gums, gelatin, acacia, and microcrystalline cellulose in amounts up to about 20% and preferably from about 1% to about 15% by weight of the pharmaceutical composition.

[0208] Sweeteners which may be employed include those sweeteners, both natural and artificial, well known in the art. Sweetening agents such as monosaccharides, disaccharides and polysaccharides such as xylose, ribose, glucose, mannose, galactose, fructose, dextrose, sucrose, maltose, partially hydrolyzed starch or corn syrup solids and sugar alcohols such as sorbitol, xylitol, mannitol and mixtures thereof may be utilized in amounts from about 10% to about 60% and preferably from about 20% to about 50% by weight of the pharmaceutical composition. Water soluble artificial sweeteners such as saccharin and saccharin salts such as sodium or calcium, cyclamate salts, acesulfame-K, aspartame and the like and mixtures thereof may be utilized in amounts from about 0.001% to about 5% by weight of the composition.

[0209] Flavorants which may be employed in the pharmaceutical products of the invention include both natural and artificial flavors, and mints such as peppermint, menthol, vanilla, artificial vanilla, chocolate, artificial chocolate, cinnamon, various fruit flavors, both individually and mixed, in amounts from about 0.5% to about 5% by weight of the pharmaceutical composition.

[0210] Colorants useful in the present invention include pigments which may be incorporated in amounts of up to about 6% by weight of the composition. A preferred pigment, titanium dioxide, may be incorporated in amounts up to about 1%. Also, the colorants may include other dyes suitable for food, drug and cosmetic applications, known as F.D.&C. dyes and the like. Such dyes are generally present in amounts up to about 0.25% and preferably from about 0.05% to about 0.2% by weight of the pharmaceutical composition. A full recitation of all F.D.&C. and D.&C. dyes and their corresponding chemical structures may be found in the Kirk-Othmer Encyclopedia of Chemical Technology, in Volume 5, at pages 857-884, which text is accordingly incorporated herein by reference.

[0211] Useful solubilizers include alcohol, propylene glycol, polyethylene glycol and the like and may be used to solubilize the flavors. Solubilizing agents are generally present in amounts up to about 10%; preferably from about 2% to about 5% by weight of the pharmaceutical composition.

[0212] Lubricating agents which may be used when desired in the instant compositions include silicone oils or fluids such as substituted and unsubstituted polysiloxanes, e.g., dimethyl polysiloxane, also known as dimethicone. Other well known lubricating agents may be employed.

[0213] It is not expected that compounds of the present invention will display significant adverse interactions with other synthetic or naturally occurring substances. Thus, a compound of the present invention may be administered in combination with other compounds and compositions. In particular the compounds of the present invention may be administered in combination with other compounds of the present invention and other targeted nuclear transcription elements substances.

[0214] The optimal pharmaceutical formulations will be determined by one skilled in the art depending upon considerations such as the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present therapeutic agents of the invention.

Route(s) of Administration

[0215] The route(s) of administration of the compounds and compositions of the present invention are well known to those skilled in the art (see, for example, “Remington's Pharmaceutical Sciences”, 18th Edition, Chapter 86, pp. 1581-1592, Mack Publishing Company, 1990). The compounds and compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneally, intrathecally, intraventricularly, intrasternal, and intracranial injection or infusion techniques.

[0216] The compounds and compositions may be administered in the form of sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions. These suspensions, may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil such as a synthetic mono- or di-glyceride may be employed. Fatty acids such as oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0217] Additionally, the compounds and compositions may be administered orally in the form of capsules, tablets, aqueous suspensions, or solutions. Tablets may contain carriers such as lactose and corn starch, and/or lubricating agents such as magnesium stearate. Capsules may contain diluents including lactose and dried corn starch. Aqueous suspensions may contain emulsifying and suspending agents combined with the active ingredient. The oral dosage forms may further contain sweetening, flavoring, coloring agents, or combinations thereof. Delivery in an enterically coated tablet, caplet, or capsule, to further enhance stability and provide release in the intestinal tract to improve absorption, is the best mode of administration currently contemplated.

[0218] The compounds may also be administered rectally in the form of suppositories. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature, but liquid at rectal temperature and, therefore, will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

[0219] Furthermore, the compounds may be administered topically, especially when the conditions addressed for treatment involve areas or organs readily accessible by topical application, including the lower intestinal tract. Suitable topical formulations can be readily prepared for such areas or organs. For example, topical application to the lower intestinal tract can be effected in a rectal suppository formulations (see above) or in suitable enema formulations.

[0220] It is envisioned that the continuous administration or sustained delivery of the compounds and compositions of the present invention may be advantageous for a given condition. While continuous administration may be accomplished via a mechanical means, such as with an infusion pump, it is contemplated that other modes of continuous or near continuous administration may be practiced. For example, such administration may be by subcutaneous or muscular injections as well as oral pills.

[0221] Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible particles or beads and depot injections, are also known to those skilled in the art.

Dosage

[0222] Dosage levels of chimeric proteins and nucleic acid chimeras are known to those of ordinary skill in the art. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

[0223] It is understood, however, that a specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; drug combination; the severity of the particular disorder being treated; and the form of administration. One of ordinary skill in the art would appreciate the variability of such factors and would be able to establish specific dose levels using no more than routine experimentation.

EXAMPLES

[0224] The following examples are illustrative of the present invention and are not intended to be limitations thereon. Unless otherwise indicated, all percentages are based upon 100% by weight of the final composition.

Example 1

[0225] Production of a GFP-GR-RAR Chimera

[0226] The 1471.1 cell line was used for this transfection study. Cells were maintained in DMEM with 10% characterized fetal calf serum, L-glutamine, and antibiotics. For transient transfections, 2×10⁵ cells were resuspended in cold DMEM without serum with the indicated plasmids and incubated on ice for 1 minute. Electroporations were done in an electroporator at 140 V, 3 pulses and 10 msec pulse length followed by a 10 minute recovery on ice. Cells were then plated and allowed to recover overnight in complete media.

[0227] Receptor Chimera. Sequences 3′ to the coding region for helix 1 of the LBD of GFP-tagged rat GR were replaced by a multiple cloning site polylinker as follows: a PCR fragment was generated to contain GR sequences including a unique Sph I site and extending through the end of the LBD H1 with the polylinker fused at the 3′ end, using suitable 5′ and 3′ primers. The PCR product was restricted with Sph I and Xba I and inserted at the corresponding Sph I and Xba I sites in pCI-nGFP-C656G. A version tagged with EGFP was also made by cloning the Nhe I-filled in BspE I fragment from EGFP-C1 between the Nhe I and filled in BssH II sites of the LBD-deleted C656G GR. The LBD-deleted C656G GR constructs were confirmed by sequencing.

[0228] Different GR-RAR chimeras were generated by cloning portions of the LBD from a human RAR-alpha cDNA in-frame into the multiple cloning site polylinker of LBD-deleted GR. The chimera shown in FIG. 1 was made by fusing the RAR-LBD, starting at the end of its H1 coding region, from amino acid residue 195, and extending through the end of coding sequence and 300 bases of 3′ untranslated region, to the end of the GR LBD helix 1 region. The RAR-LBD-containing fragment from filled in Fsp I to Eco RI was cloned between the filled in Xho I and the Eco RI sites of LBD-deleted C656G GR, and was confirmed by sequencing.

[0229] Fluorescence Microscopy. For fluorescence microscopy, cells were electroporated with 2 ug GFP-GR-RAR, GFP-GR, or GFP-RAR. After electroporation, {fraction (1/24)}th of the cells were plated in each well of a 6-well dish onto a clean #1.5 glass coverslip in DMEM containing 10% charcoal/dextran-treated FCS. The next day, cells were treated with the indicated ligand for 1 hour, cells on coverslips were rinsed with PBS, inverted onto microscope slides, and immediately viewed on a scanning confocal microscope. GFP was excited with a 488 nm laser lines supplied from a air cooled argon laser. GFP emission was monitored between 505 and 590 nm with a pinhole setting of 1.0.

[0230] Assay of Receptor Activity. Cells were electroporated with 5 ug pLTRLuc, containing the MMTV LTR, and 0.7 ug pCMV-betaGal with or without 10 ug of the indicated receptor expression vector as described above. Cells were plated in duplicate in 100 mm dishes in DMEM containing 10% charcoal/dextran-treated FCS, as described above, and allowed to recover overnight. The next day, cells were treated with the indicated ligand for 6 hours. As shown in FIG. 2, luciferase and beta-galactosidase were assayed.

[0231] Co-immunoprecipitation Assays. Transfected cells were harvested at 36-48 hrs and lysed in 300 ul of HEGM+0.5% NP40+2 mM DTT per 107 cells for 5 min at 40° C., where HEGM is 20 mM Hepes pH7, 1 mM EDTA, 10% glycerol, 10 mM Sodium Molybdate. A protease inhibitor cocktail was also included. Nuclei were pelleted and the cytoplasmic extract was precleared with blank protein G sepharose for 2 hrs at 40° C. and then immunoadsorbed against protein G sepharose pre-loaded with anti-GFP monoclonal antibody overnight at 40° C. Sepharose pellets were then washed with 3×1 ml of HEM+50 mM NaCl+0.5% Tween 20, boiled in electrophoresis loading buffer, run on 4-12% Bis-Tris gradient gels and blotted. Western blots were sequentially probed first with anti-HSP90 monoclonal antibody, then with anti-GFP antibody, and finally with anti-GR antipeptide polyclonal antibody.

[0232] Results. Applicants have modified the ligand binding domain (LBD) of GFP-GR in such a way that the unique cytoplasmic/nuclear translocation activity of GR was maintained, but the modified chimera responded to a superfamily receptor ligand.

[0233] Subcellular trafficking was examined in response to ligands. Representative results are shown in FIG. 3; in each case, essentially all of the transfected cells in culture responded uniformly in the same manner as the examples shown. The GR receptor was completely cytoplasmic in the absence of ligand and became exclusively nuclear in response to dexamethasone, as shown in FIG. 3, panels D and E but was unaffected by ATRA as shown in FIG. 3, panel F. As expected, wild type RAR was always nuclear, as shown in FIG. 3, panels G-I. GR-RAR was cytoplasmic in non-treated cells, remained cytoplasmic after dexamethasone treatment, as shown in FIG. 3, panels A and B, but translocated to the nucleus with ATRA, as shown in FIG. 3, panel C. That is, the GR-RAR chimera retained the cytoplasmic localization of ligand-free wild-type GR, but translocated in response to the normal RAR ligand rather than GR agonists.

[0234] We believed that HSP90 binding by GR is important to a properly folded protein structure. Accordingly, the ability of the chimeric GR-RAR receptor to interact with HSP90 was preserved with the new LBD.

[0235] Since we also believed that the cytoplasmic interaction with HSP90 was critical for steroid binding and regulated translocation of the receptor, it was of interest to determine whether the ability of the chimeric GR-RAR receptor to interact with HSP90 was retained. Cytoplasmic extracts from cells transfected with GR, the GR-RAR chimera fused to GFP, or with native EGFP, were each immunoprecipitated with anti-GFP antibody and the precipitates were probed for the presence of bound HSP90, as shown in FIG. 4. Both cytoplasmic GR and GR-RAR chimeras interacted specifically with HSP90, while EGFP did not, demonstrating that HSP90 interactions key to ligand-regulated translocation were conserved in the chimeric protein.

[0236]FIG. 1 is a drawing which depicts the construction of a chimeric GFP-GR-RAR receptor. A GFP-tagged version of rat GR was modified with the insertion of a polylinker at amino acid position 570, replacing the LBD. A C-terminal fragment of the RAR receptor beginning at amino acid position 195 was fused to GFP-GR at an Xho I site in the polylinker. The resulting fusion protein includes the last six amino acids of the RAR LBD helix 1, as well as the complete GR LBD helix 1. Another GR domain included in the fusion is the domain required for HSP90 binding.

[0237]FIG. 2 is a graph which depicts transcriptional transactivation by the chimeric receptor. Transfected cells were treated with the indicated ligand for 6 hours. Fold inductions are shown in the bottom table, and were calculated from the luciferase activity, normalized to beta-galactosidase activity, of ligand induced samples compared to untreated controls. Experiments were carried out twice in duplicate and a representative example is shown for mock transfected cells control cells transfected with a GFP-GR C656G mutant “super” receptor GR*, and cells transfected with the GR-RAR chimeras. The data in Table 1 shows that the chimeric GR-RAR protein of the present invention is induced by all-trans retinoic acid, but not physiologically relevant concentrations of dexamethasone, and the chimeric protein translocated in the presence of its LBD ligand, ATRA. TABLE 1 Transcriptional Activation of Control and Chimeric Proteins Control (Endogenous GR) GR* GR-RAR Control 1.00 1.00 1.00 1 nM Dex 1.35 17.4 1.33 100 nM ATRA 1.27 1.78 18.6

[0238]FIG. 3 is a series of photographs which depict subcellular trafficking of chimeric receptor. Mouse mammary adenocarcinoma cells were transfected with GFP-GR-RAR (panels A, B, C), GFP-GR (panels D, E, F), or GFP-RAR (panels G, H, I). Cells were treated for 1 hour with medium containing only charcoal stripped-serum (panels A, D, G), or 100 nM dexamethasone (panels B, E, H), or 100 nM all-trans-retinoic acid (panels C, F, I). The cell outline is indicated by a dashed white line in panels C, E, G, H, and I, the examples in which the subcellular distribution is nuclear. The cells shown in panels D and I are binucleate.

[0239]FIG. 4 is a photograph which depicts the interaction of the chimeric receptor with heat shock protein 90. Cytoplasmic extracts from cells transfected with GFP fusions to GR* or to GR-RAR, or with native EGFP, were immunoprecipitated using an anti-GFP monoclonal antibody, separated on a 4-12% gradient gel, blotted, and probed with anti-HSP90 antibody. About 0.5% of each extract used prior to immunoprecipitation was also run for comparison. Blots were subsequently reprobed with anti-GFP and with anti-GR antibodies as a control to confirm uniformity in binding and recovery of immunoprecipitate. The anti-GFP antibody used for western blots reacted poorly with the GFP-fusion proteins compared to unmodified EGFP and was undetectable in the load. Anti-GR was used as a second control for binding and recovery. FIG. 4 shows that the GFP-GR-RAR chimeric protein binds to HSP90 with similar affinity to GR-HSP90 binding.

[0240] For additional discussion of the procedures followed and results obtained in Example 1, Applicants' publication, Mackem S, Baumann C T, Hager G L, A glucocorticoid/retinoic acid receptor chimera that displays cytoplasmic/nuclear translocation in response to retinoic acid. A real time sensing assay for nuclear receptor ligands, J Biol Chem. 276(49):45501-4 (2001), is incorporated by reference in its entirety.

Example 2

[0241] Production of GFP-GR-ER Chimera

[0242] Using techniques substantially similar to those described above in Example 1, Applicants have produced a GFP-GR-ER DNA chimera, which translates to a GFP-GR-ER chimeric protein which exhibits the following characteristics: (1) within the limits of detection, complete isolation of the chimeric protein in the cytoplasm of a cell in the absence of ligand, estrogen, for its LBD, and rapid, essentially complete translocation to the nucleus in the presence of estrogen at a concentration of 100 nM.

[0243] Thus, FIG. 5 depicts the construction of a chimeric GFP-GR-ER receptor. A GFP-tagged version of rat GR was modified with the insertion of a polylinker at amino acid position 570, replacing the LBD. A C-terminal fragment of a GFP-tagged ER receptor, beginning at amino acid position 323 as correlated to the ER sequence published as Accession No. NM000125, was fused to GFP-GR at a Stu I/Blp I site in the polylinker. The resulting fusion protein includes part of the ER LBD 1-3 loop as a spacer, as well as the complete GR LBD helix 1. Another GR domain included in the chimera is the heptapeptide element essential for HSP90 binding.

[0244]FIG. 6 is a series of photographs which depict subcellular trafficking of wild type ER receptor, without estrogen (two panels A and B) and in the presence of estrogen at 100 nM concentration (panels C and D). The cell outline is indicated by a dashed white line in all panels. As is clearly shown, wild type ER is distributed throughout the cell nucleus with or without ligand.

[0245]FIG. 7 is a series of photographs which depict subcellular trafficking of a chimeric GFP-GR-ER receptor, without estrogen (panels A, B, and C) and in the presence of estrogen at 100 nM concentration (panels D, E, and F). The cell outline is indicated by a dashed white line in panels D, E, and F. The cells shown in panel D is binucleate. As is clearly shown, chimeric GFP-GR-ER receptor is distributed exclusively in the cell cytoplasm without ligand, and translocates to exclusive distribution in the nucleus in the presence of ligand.

Example 3 Production of Additional GFP-GR-Receptor Chimeras

[0246] Almost all superfamily receptors share a highly conserved zinc-finger DNA-binding domain (DBD) and a less conserved ligand-binding domain (LBD). Pre-genome methods previously identified 48 human nuclear receptor genes. A recent study of the sequence of the human genome found all 48 known nuclear receptors, including 48 LBDs, and estimates that there are about 75 superfamily receptors. Table 2 is a catalogue of the known genes coding for superfamily receptor proteins. TABLE 2 Candidate Receptor Proteins for GR Chimeras GenBank Gene accession nomenclature Localization number Domain(s) identified TRα NR1A1 chr. 17 AC068669 DBD + LBD TRβ NR1A2 chr. 3 AC018451.11 DBD + LBD RARα NR1B1 chr. 17 AC018629 DBD + LBD RARβ NR1B2 chr. 3 AC011323 DBD chr. 3.332 AC012037.11 LBD RARγ NR1B3 chr. 12 AC073573 DBD + LBD PPARα chr. 22.431 AL078611.1 DBD + begining-LBD chr. 22.432 AL078611.1 end-LBD PPARβ NR1C2 chr. 6 AL022721 DBD + LBD PPARγ NR1C3 chr. 3.184 AC016333.5 DBD + LBD Rev-erbα NR1D1 chr. 17 AC068669 DBD + LBD Rev-erbβ NR1D2 chr. 3.317 AC046128.6 LBD orphan rev-erb chr. 2.1230 AC012444.4 mid-LBD fragment RORα NR1F1 chr. 15.575 AC012344 DBD + LBD RORβ NR1F2 chr. 9 AL137018 DBD + LBD RORγ NR1F3 chr. 1.1727 AC068971.2 DBD + LBD LXRα NR1H2 chr. 11.503 AC018410 DBD + LBD LXRβ NR1H3 chr. 19 AC073646 DBD + LBD FXR NR1H4 chr. 12.1078 AC010200.7 DBD chr. 12.1079 AC010200.7 end-DBD + LBD FXRβ NR1H5 chr. 1 AL390235 DBD + partial-LBD chr. 1 AL138783 LBD VDR NR1I1 chr. 12.502 AC004466 DBD + LBD PXR NR1I2 chr. 3.1354 AC069444 DBD + LBD CAR NR1I3 chr. 1 AL590714 DBD + LBD HNF4a NR2A1 chr. 20.444 AL132772 DBD + LBD HNF4g NR2A2 chr. 8.827 AC061989.2 DBD + LBD pseudo-HNF4g chr. 13 AL158054 DBD + LBD RXRα NR2B1 chr. 9 AL158031 DBD + LBD RXRβ NR2B2 chr. 6.367 AL031228.1 DBD + LBD RXRγ NR2B3 chr. 1.1870 AC009625.4 DBD + partial-LBD TR2 NR2C1 chr. 12 AC011598 DBD + LBD TR4 NR2C2 chr. 3.227 AC011699.9 DBD + LBD TLL NR2E1 chr. 6.1168 AC027711.2 DBD + LBD PNR NR2E3 chr. 7 AC084299 DBD + LBD COUPα NR2F1 chr. 5.1035 AC008516 DBD + LBD COUPβ NR2F2 chr. 15.948 AC016251 DBD + LBD EAR2 NE2F6 chr. 14 AC010646 DBD + LBD pseudo-EAR2 chr. 15.175 AC020679 mid-LBD ERα NR3A1 chr. 6.1623 AC058817.4 DBD chr. 6.1624 AL049821.6 mid-LBD chr. 6.1625 AL078582.13 end-LBD ERβ NR3A2 chr. 14 CNS01RHJ/ DBD + LBD AL161756 ERRα NR3B1 chr. 11.683 AC005848.1 DBD + partial-LBD pseudo-ERRα chr. 13.164 AC021256.4 DBD + LBD ERRβ NR3B2 chr. 14.757 AC016543.5 DBD + begining-LBD chr. 14.758 AC008050.6 LBD ERRγ NR3B3 chr. 1 AL356008 DBD + partial LBD chr. 1 AL512650 LBD GR NR3C1 chr. 5.1617 AC012634.6 DBD + LBD MR NR3C2 chr. 4.1548 AC010833.3 DBD + begining-LBD PR NR3C3 chr. 11 AP001533 DBD + LBD AR NR3C4 chr. X.615 AL158016.9 begining-DBD chr. X.616 AL158016.9 end-DBD + LBD NGFIB NR4A1 chr. 12.549 AC019244.3 DBD + LBD NURR1 NR4A2 chr. 2.1627 AC073847.1 DBD + LBD NOR1 NR4A3 chr. 9.1023 AC073459.3 DBD + LBD SF1 NR5A1 chr. 9.1275 AL137846 DBD + LBD LRH1 NR5A2 chr. 1.2296 AF190464 DBD + begining-LBD chr. 1.2297 AF190464.1 mid-LBD chr. 1.2298 AC024348.3 end-LBD GCNF1 NR6A1 chr. 9.1275 AL354979 DBD + LBD SHP NR0B3 chr. 1.294 AC004873.2 LBD DAX NR0B1 chr. X AC005185 LBD

[0247] The above identification of superfamily receptors having DBDs and LDBs by the Human Genome Project provide candidate receptors for the methods of the present invention. It is expected that particularly those genes with no known ligand may be analyzed by the inventive methods to quickly and cheaply screen candidate ligands and ligand analogues.

[0248]FIG. 8 is a chart which depicts sequence alignments of the protein sequence surrounding helix 1 and helix 3 in several representative mammalian steroid receptors. The sequences in the region of helix 3 demonstrate a relatively high degree of sequence homology, especially when taking into consideration conservative amino acid substitutions. Further, there is even greater functional homology in these domains, given the known and predicted helical tertiary structures in the putative helix 1 and helix 3 domains.

[0249] Considering the classic steroid receptor amino sequences shown in FIG. 9, which depicts sequence alignments of the protein sequences surrounding helix 3, it is expected that chimeric molecules may be synthesized from all of these receptors.

[0250]FIG. 10 depicts the synthetic strategy for the synthesis of chimeric GFP-GR-receptor DNA sequences and the corresponding proteins. A GR polypeptide fragment, complete through the DBD, the translocation sequence, and Helix 1, and a linker, such as a multiple cloning site polylinker, are expected to be included in the chimeras. In addition, the LBD, the complete Helix 3 of the desired receptor, and a 5′ linker, for example the ER 1-3 loop, are also expected to be included in the chimeras.

[0251] Applicants work indicates that a linker region which is too short, for example by omitting a 5′ linker from the receptor fragment, reduces, but does not eliminate, the translocation efficiency of a chimera. On the other hand, inclusion of a second Helix 1 in the RAR receptor fragment of the chimera, as occurred in GFP-GR-RAR chimera H1 shown in FIG. 10, appears to eliminate the translocation characteristics of that recombinant protein. It is expected that this effect on translocation is a result of changes in the tertiary structure of the recombinant protein produced by a linker region which is simply too long or a result of improper folding of the recombinant protein produced by the presence of the second Helix 1.

[0252] As is evident from the present successful results, it is expected that we will successfully generate similar DNA chimeras and corresponding chimeric proteins using other receptor LBDs, and then to identify new ligands and to detect ligand levels in situ. Such chimeras provide a ligand detection tool fundamentally distinct from existing techniques, which test receptor responses at the gene activation, or transcriptional, level. The approach described here tests directly for the ability of an LBD to induce translocation and hence interactions with transcriptional coactivators, chromatin remodeling factors, etc, are unlikely to play a significant role.

Example 4

[0253] Direct Visualization Assays of Ligand Distribution in Living Cells, in Real Time

[0254] A GFP-coupled GR-receptor chimera, such as GFP-GR-ER or GFP-GR-RAR, provides the unique capability to directly visualize dynamic, temporo-spatial distributions of superfamily receptors in real time, in live organisms. A GFP-GR-RAR protein is a representative example.

[0255] Localization efforts using retinoid-responsive promoters to drive reporter genes are inherently limited by the slow response and level of sensitivity to ligand in co-cultures of reporter cells with tissue explants, or by their dependence on endogenous cellular receptors, cofactors, and the limitations of the particular transgenic promoters used. In contrast, the inventive translocation based assay requires only about 20-30 minutes of exposure, is visualized directly in vivo, and is independent of the particular transcriptional milieu of the cell.

[0256] Screens for novel receptor ligands using transcriptional reporters were previously similarly limited, whereas the translocation assay with a GFP-tagged chimera requires no biochemical manipulations and allows rapid, direct visualization in living cells. Epifluorescent cellular imaging systems driven by artificial intelligence software can now automatically scan and quantify the subcellular distribution of fluorescent molecules in large numbers of living cell pools. These imaging systems, coupled with the inventive translocation assay, can be employed in high-throughput screens for novel receptor ligands in vivo.

[0257] It is expected that the inventive translocation assays will provide equal or better sensitivity, at lower cost, with higher throughput rates, and with quicker processing times when compared to other assays known in the art.

[0258] The invention being thus described, it will be obvious that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications and variations are intended to be included within the scope of the following claims.

1 6 1 1147 PRT Rat/human chimera mat_peptide (1)..() Chimeric Protein 1 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Gly Ala 225 230 235 240 Gly Ala Gly Ala Gly Ala Gly Ala Ile Ser Ala Leu Leu Asp Ser Lys 245 250 255 Glu Ser Leu Ala Pro Pro Gly Arg Asp Glu Val Pro Gly Ser Leu Leu 260 265 270 Gly Gln Gly Arg Gly Ser Val Met Asp Phe Tyr Lys Ser Leu Arg Gly 275 280 285 Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Val Ala Ala Ala 290 295 300 Ser Gln Ala Asp Ser Lys Gln Gln Arg Ile Leu Leu Asp Phe Ser Lys 305 310 315 320 Gly Ser Thr Ser Asn Val Gln Gln Arg Gln Gln Gln Gln Gln Gln Gln 325 330 335 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Gly Leu Ser 340 345 350 Lys Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu Thr 355 360 365 Lys Val Met Gly Asn Asp Leu Gly Tyr Pro Gln Gln Gly Gln Leu Gly 370 375 380 Leu Ser Ser Gly Glu Thr Asp Phe Arg Leu Leu Glu Glu Ser Ile Ala 385 390 395 400 Asn Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser Thr 405 410 415 Ser Ala Thr Gly Cys Ala Thr Pro Thr Glu Lys Glu Phe Pro Lys Thr 420 425 430 His Ser Asp Ala Ser Ser Glu Gln Gln Asn Arg Lys Ser Gln Thr Gly 435 440 445 Thr Asn Gly Gly Ser Val Lys Leu Tyr Pro Thr Asp Gln Ser Thr Phe 450 455 460 Asp Leu Leu Lys Asp Leu Glu Phe Ser Ala Gly Ser Pro Ser Lys Asp 465 470 475 480 Thr Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn Leu 485 490 495 Leu Ser Pro Leu Ala Gly Glu Asp Asp Pro Phe Leu Leu Glu Gly Asn 500 505 510 Thr Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro Lys 515 520 525 Ile Lys Asp Thr Gly Asp Thr Ile Leu Ser Ser Pro Ser Ser Val Ala 530 535 540 Leu Pro Gln Val Lys Thr Glu Lys Asp Asp Phe Ile Glu Leu Cys Thr 545 550 555 560 Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Pro Val Tyr Cys Gln Ala 565 570 575 Ser Phe Ser Gly Thr Asn Ile Ile Gly Asn Lys Met Ser Ala Ile Ser 580 585 590 Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp Met 595 600 605 Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Val Phe Asn 610 615 620 Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys Gln 625 630 635 640 Gly Ser Gly Glu Asp Ser Leu Thr Ser Leu Gly Ala Leu Asn Phe Pro 645 650 655 Gly Arg Ser Val Phe Ser Asn Gly Tyr Ser Ser Pro Gly Met Arg Pro 660 665 670 Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Ala Ala Thr Gly Pro Pro 675 680 685 Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His Tyr 690 695 700 Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala Val 705 710 715 720 Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile Ile 725 730 735 Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys Cys 740 745 750 Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys Ile 755 760 765 Lys Gly Ile Gln Gln Ala Thr Ala Gly Val Ser Gln Asp Thr Ser Glu 770 775 780 Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu Pro Gln Leu Thr Pro 785 790 795 800 Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu Tyr Ala 805 810 815 Gly Tyr Asp Ser Ser Ser Arg Gly Glu Pro Pro Ile Leu Tyr Ser Glu 820 825 830 Tyr Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly Leu Leu 835 840 845 Thr Asn Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Ser 850 855 860 Arg Gly Leu Ser Ser Lys Ala His Gln Glu Thr Phe Pro Ala Leu Cys 865 870 875 880 Gln Leu Gly Lys Tyr Thr Thr Asn Asn Ser Ser Glu Gln Arg Val Ser 885 890 895 Leu Asp Ile Asp Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp 900 905 910 Ala Lys Arg Val Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln Val 915 920 925 His Leu Leu Glu Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val 930 935 940 Trp Arg Ser Met Glu His Pro Val Lys Leu Leu Phe Ala Pro Asn Leu 945 950 955 960 Leu Leu Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile 965 970 975 Phe Asp Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu 980 985 990 Gln Gly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser 995 1000 1005 Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu 1010 1015 1020 Lys Asp His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu 1025 1030 1035 Ile His Leu Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His 1040 1045 1050 Gln Arg Leu Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His 1055 1060 1065 Met Ser Asn Lys Gly Met Glu His Leu Tyr Ser Met Lys Cys Lys 1070 1075 1080 Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala 1085 1090 1095 His Arg Leu His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val Glu 1100 1105 1110 Glu Thr Asp Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser 1115 1120 1125 His Ser Leu Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe 1130 1135 1140 Pro Ala Thr Val 1145 2 1089 PRT Rat/human chimera mat_peptide (1)..() Chimeric Protein 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 Gly Ala Leu Leu Asp Ser Lys Glu Ser Leu Ala Pro Pro Gly Arg Asp 245 250 255 Glu Val Pro Gly Ser Leu Leu Gly Gln Gly Arg Gly Ser Val Met Asp 260 265 270 Phe Tyr Lys Ser Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala Ser 275 280 285 Ser Pro Ser Val Ala Ala Ala Ser Gln Ala Asp Ser Lys Gln Gln Arg 290 295 300 Ile Leu Leu Asp Phe Ser Lys Gly Ser Thr Ser Asn Val Gln Gln Arg 305 310 315 320 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 325 330 335 Gln Gln Gln Pro Gly Leu Ser Lys Ala Val Ser Leu Ser Met Gly Leu 340 345 350 Tyr Met Gly Glu Thr Glu Thr Lys Val Met Gly Asn Asp Leu Gly Tyr 355 360 365 Pro Gln Gln Gly Gln Leu Gly Leu Ser Ser Gly Glu Thr Asp Phe Arg 370 375 380 Leu Leu Glu Glu Ser Ile Ala Asn Leu Asn Arg Ser Thr Ser Val Pro 385 390 395 400 Glu Asn Pro Lys Ser Ser Thr Ser Ala Thr Gly Cys Ala Thr Pro Thr 405 410 415 Glu Lys Glu Phe Pro Lys Thr His Ser Asp Ala Ser Ser Glu Gln Gln 420 425 430 Asn Arg Lys Ser Gln Thr Gly Thr Asn Gly Gly Ser Val Lys Leu Tyr 435 440 445 Pro Thr Asp Gln Ser Thr Phe Asp Leu Leu Lys Asp Leu Glu Phe Ser 450 455 460 Ala Gly Ser Pro Ser Lys Asp Thr Asn Glu Ser Pro Trp Arg Ser Asp 465 470 475 480 Leu Leu Ile Asp Glu Asn Leu Leu Ser Pro Leu Ala Gly Glu Asp Asp 485 490 495 Pro Phe Leu Leu Glu Gly Asn Thr Asn Glu Asp Cys Lys Pro Leu Ile 500 505 510 Leu Pro Asp Thr Lys Pro Lys Ile Lys Asp Thr Gly Asp Thr Ile Leu 515 520 525 Ser Ser Pro Ser Ser Val Ala Leu Pro Gln Val Lys Thr Glu Lys Asp 530 535 540 Asp Phe Ile Glu Leu Cys Thr Pro Gly Val Ile Lys Gln Glu Lys Leu 545 550 555 560 Gly Pro Val Tyr Cys Gln Ala Ser Phe Ser Gly Thr Asn Ile Ile Gly 565 570 575 Asn Lys Met Ser Ala Ile Ser Val His Gly Val Ser Thr Ser Gly Gly 580 585 590 Gln Met Tyr His Tyr Asp Met Asn Thr Ala Ser Leu Ser Gln Gln Gln 595 600 605 Asp Gln Lys Pro Val Phe Asn Val Ile Pro Pro Ile Pro Val Gly Ser 610 615 620 Glu Asn Trp Asn Arg Cys Gln Gly Ser Gly Glu Asp Ser Leu Thr Ser 625 630 635 640 Leu Gly Ala Leu Asn Phe Pro Gly Arg Ser Val Phe Ser Asn Gly Tyr 645 650 655 Ser Ser Pro Gly Met Arg Pro Asp Val Ser Ser Pro Pro Ser Ser Ser 660 665 670 Ser Ala Ala Thr Gly Pro Pro Pro Lys Leu Cys Leu Val Cys Ser Asp 675 680 685 Glu Ala Ser Gly Cys His Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys 690 695 700 Val Phe Phe Lys Arg Ala Val Glu Gly Gln His Asn Tyr Leu Cys Ala 705 710 715 720 Gly Arg Asn Asp Cys Ile Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro 725 730 735 Ala Cys Arg Tyr Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala 740 745 750 Arg Lys Thr Lys Lys Lys Ile Lys Gly Ile Gln Gln Ala Thr Ala Gly 755 760 765 Val Ser Gln Asp Thr Ser Glu Asn Pro Asn Lys Thr Ile Val Pro Ala 770 775 780 Ala Leu Pro Gln Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val Ile 785 790 795 800 Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Ser Arg Gly 805 810 815 Leu Ser Ser Lys Ala His Gln Glu Thr Phe Pro Ala Leu Cys Gln Leu 820 825 830 Gly Lys Tyr Thr Thr Asn Asn Ser Ser Glu Gln Arg Val Ser Leu Asp 835 840 845 Ile Asp Leu Trp Asp Lys Phe Ser Glu Leu Ser Thr Lys Cys Ile Ile 850 855 860 Lys Thr Val Glu Phe Ala Lys Gln Leu Pro Gly Phe Thr Thr Leu Thr 865 870 875 880 Ile Ala Asp Gln Ile Thr Leu Leu Lys Ala Ala Cys Leu Asp Ile Leu 885 890 895 Ile Leu Arg Ile Cys Thr Arg Tyr Thr Pro Glu Gln Asp Thr Met Thr 900 905 910 Phe Ser Asp Gly Leu Thr Leu Asn Arg Thr Gln Met His Asn Ala Gly 915 920 925 Phe Gly Pro Leu Thr Asp Leu Val Phe Ala Phe Ala Asn Gln Leu Leu 930 935 940 Pro Leu Glu Met Asp Asp Ala Glu Thr Gly Leu Leu Ser Ala Ile Cys 945 950 955 960 Leu Ile Cys Gly Asp Arg Gln Asp Leu Glu Gln Pro Asp Arg Val Asp 965 970 975 Met Leu Gln Glu Pro Leu Leu Glu Ala Leu Lys Val Tyr Val Arg Lys 980 985 990 Arg Arg Pro Ser Arg Pro His Met Phe Pro Lys Met Leu Met Lys Ile 995 1000 1005 Thr Asp Leu Arg Ser Ile Ser Ala Lys Gly Ala Glu Arg Val Ile 1010 1015 1020 Thr Leu Lys Met Glu Ile Pro Gly Ser Met Pro Pro Leu Ile Gln 1025 1030 1035 Glu Met Leu Glu Asn Ser Glu Gly Leu Asp Thr Leu Ser Gly Gln 1040 1045 1050 Pro Gly Gly Gly Gly Arg Asp Gly Gly Gly Leu Ala Pro Pro Pro 1055 1060 1065 Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser Ser Asn Arg Ser Ser 1070 1075 1080 Pro Ala Thr His Ser Pro 1085 3 3300 DNA Rat/human Chimera CDS (1)..(3300) 3 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa tta gat ggt gat gtt aat ggg cac aaa ttt tct gtc agt gga gag 96 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act act gga aaa cta cct gtt cct tgg cca aca ctt gtc act act ttc 192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 act tat ggt gtt caa tgc ttt tca aga tac cca gat cat atg aaa cag 240 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt ttc aaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa ttg gaa tac aac 432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 tat aac tca cac aat gta tac atc atg gca gac aaa caa aag aat gga 480 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaa att aga cac aac att gaa gat gga agc gtt 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 caa cta gca gac cat tat caa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aac cat tac ctg tcc aca caa tct gcc ctt tcg 624 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aag aga gac cac atg gtc ctt ctt gag ttt gta 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa ggc gcc 720 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Gly Ala 225 230 235 240 ggc gct ggt gct ggt gct ggc gcc atc agc gcg ctg atc ctg gac tcc 768 Gly Ala Gly Ala Gly Ala Gly Ala Ile Ser Ala Leu Ile Leu Asp Ser 245 250 255 aaa gaa tcc tta gct ccc cct ggt aga gac gaa gtc cct ggc agt ttg 816 Lys Glu Ser Leu Ala Pro Pro Gly Arg Asp Glu Val Pro Gly Ser Leu 260 265 270 ctt ggc cag ggg agg ggg agc gta atg gac ttt tat aaa agc ctg agg 864 Leu Gly Gln Gly Arg Gly Ser Val Met Asp Phe Tyr Lys Ser Leu Arg 275 280 285 gga gga gct aca gtc aag gtt tct gca tct tcg ccc tca gtg gct gct 912 Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Val Ala Ala 290 295 300 gct tct cag gca gat tcc aag cag cag agg att ctc ctt gat ttc tcg 960 Ala Ser Gln Ala Asp Ser Lys Gln Gln Arg Ile Leu Leu Asp Phe Ser 305 310 315 320 aaa ggc tcc aca agc aat gtg cag cag cga cag cag cag cag cag cag 1008 Lys Gly Ser Thr Ser Asn Val Gln Gln Arg Gln Gln Gln Gln Gln Gln 325 330 335 cag cag cag cag cag cag cag cag cag cag cag cag cag cca ggc tta 1056 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Gly Leu 340 345 350 tcc aaa gcc gtt tca ctg tcc atg ggg ctg tat atg gga gag aca gaa 1104 Ser Lys Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu 355 360 365 aca aaa gtg atg ggg aat gac ttg ggc tac cca cag cag ggc caa ctt 1152 Thr Lys Val Met Gly Asn Asp Leu Gly Tyr Pro Gln Gln Gly Gln Leu 370 375 380 ggc ctt tcc tct ggg gaa aca gac ttt cgg ctt ctg gaa gaa agc att 1200 Gly Leu Ser Ser Gly Glu Thr Asp Phe Arg Leu Leu Glu Glu Ser Ile 385 390 395 400 gca aac ctc aat agg tcg acc agc gtt cca gag aac ccc aag agt tca 1248 Ala Asn Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser 405 410 415 acg tct gca act ggg tgt gct acc ccg aca gag aag gag ttt ccc aaa 1296 Thr Ser Ala Thr Gly Cys Ala Thr Pro Thr Glu Lys Glu Phe Pro Lys 420 425 430 act cac tcg gat gca tct tca gaa cag caa aat cga aaa agc cag acc 1344 Thr His Ser Asp Ala Ser Ser Glu Gln Gln Asn Arg Lys Ser Gln Thr 435 440 445 ggc acc aac gga ggc agt gtg aaa ttg tat ccc aca gac caa agc acc 1392 Gly Thr Asn Gly Gly Ser Val Lys Leu Tyr Pro Thr Asp Gln Ser Thr 450 455 460 ttt gac ctc ttg aag gat ttg gag ttt tcc gct ggg tcc cca agt aaa 1440 Phe Asp Leu Leu Lys Asp Leu Glu Phe Ser Ala Gly Ser Pro Ser Lys 465 470 475 480 gac aca aac gag agt ccc tgg aga tca gat ctg ttg ata gat gaa aac 1488 Asp Thr Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn 485 490 495 ttg ctt tct cct ttg gcg gga gaa gat gat cca ttc ctt ctc gaa ggg 1536 Leu Leu Ser Pro Leu Ala Gly Glu Asp Asp Pro Phe Leu Leu Glu Gly 500 505 510 aac acg aat gag gat tgt aag cct ctt att tta ccg gac act aaa cct 1584 Asn Thr Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro 515 520 525 aaa att aag gat act gga gat aca atc tta tca agt ccc agc agt gtg 1632 Lys Ile Lys Asp Thr Gly Asp Thr Ile Leu Ser Ser Pro Ser Ser Val 530 535 540 gca cta ccc caa gtg aaa aca gaa aaa gat gat ttc att gaa ctt tgc 1680 Ala Leu Pro Gln Val Lys Thr Glu Lys Asp Asp Phe Ile Glu Leu Cys 545 550 555 560 acc ccc ggg gta att aag caa gag aaa ctg ggc cca gtt tat tgt cag 1728 Thr Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Pro Val Tyr Cys Gln 565 570 575 gca agc ttt tct ggg aca aat ata att ggt aat aaa atg tct gcc att 1776 Ala Ser Phe Ser Gly Thr Asn Ile Ile Gly Asn Lys Met Ser Ala Ile 580 585 590 tct gtt cat ggt gtg agt acc tct gga gga cag atg tac cac tat gac 1824 Ser Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp 595 600 605 atg aat aca gca tcc ctt tct cag cag cag gat cag aag cct gtt ttt 1872 Met Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Val Phe 610 615 620 aat gtc att cca cca att cct gtt ggt tct gaa aac tgg aat agg tgc 1920 Asn Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys 625 630 635 640 caa ggc tcc gga gag gac agc ctg act tcc ttg ggg gct ctg aac ttc 1968 Gln Gly Ser Gly Glu Asp Ser Leu Thr Ser Leu Gly Ala Leu Asn Phe 645 650 655 cca ggc cgg tca gtg ttt tct aat ggg tac tca agc cct gga atg aga 2016 Pro Gly Arg Ser Val Phe Ser Asn Gly Tyr Ser Ser Pro Gly Met Arg 660 665 670 cca gat gta agc tct cct cca tcc agc tcg tca gca gcc acg gga cca 2064 Pro Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Ala Ala Thr Gly Pro 675 680 685 cct ccc aag ctc tgc ctg gtg tgc tcc gat gaa gct tca gga tgt cat 2112 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His 690 695 700 tac ggg gtg ctg aca tgt gga agc tgc aaa gta ttc ttt aaa aga gca 2160 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala 705 710 715 720 gtg gaa gga cag cac aat tac ctt tgt gct gga aga aac gat tgc atc 2208 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile 725 730 735 att gat aaa att cga agg aaa aac tgc cca gca tgc cgc tat cgg aaa 2256 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 740 745 750 tgt ctt cag gct gga atg aac ctt gaa gct cga aaa aca aag aaa aaa 2304 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys 755 760 765 atc aaa ggg att cag caa gcc act gca gga gtc tca caa gac act tcg 2352 Ile Lys Gly Ile Gln Gln Ala Thr Ala Gly Val Ser Gln Asp Thr Ser 770 775 780 gaa aat cct aac aaa aca ata gtt cct gca gca tta cca cag ctc acc 2400 Glu Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu Pro Gln Leu Thr 785 790 795 800 cct acc ttg gtg tca ctg ctg gag gtg att gaa ccc gag gtg ttg tat 2448 Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu Tyr 805 810 815 gca gga tat gat agc tct gtt tcg cga ggt gag ccc ccc ata ctc tat 2496 Ala Gly Tyr Asp Ser Ser Val Ser Arg Gly Glu Pro Pro Ile Leu Tyr 820 825 830 tcc gag tat gat cct acc aga ccc ttc agt gaa gct tcg atg atg ggc 2544 Ser Glu Tyr Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly 835 840 845 tta ctg acc aac ctg gca gac agg gag ctg gtt cac atg atc aac tgg 2592 Leu Leu Thr Asn Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp 850 855 860 gcg aag agg gtg cca ggc ttt gtg gat ttg acc ctc cat gat cag gtc 2640 Ala Lys Arg Val Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln Val 865 870 875 880 cac ctt cta gaa tgt gcc tgg cta gag atc ctg atg att ggt ctc gtc 2688 His Leu Leu Glu Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val 885 890 895 tgg cgc tcc atg gag cac cca gtg aag cta ctg ttt gct cct aac ttg 2736 Trp Arg Ser Met Glu His Pro Val Lys Leu Leu Phe Ala Pro Asn Leu 900 905 910 ctc ttg gac agg aac cag gga aaa tgt gta gag ggc atg gtg gag atc 2784 Leu Leu Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile 915 920 925 ttc gac atg ctg ctg gct aca tca tct cgg ttc cgc atg atg aat ctg 2832 Phe Asp Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu 930 935 940 cag gga gag gag ttt gtg tgc ctc aaa tct att att ttg ctt aat tct 2880 Gln Gly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser 945 950 955 960 gga gtg tac aca ttt ctg tcc agc acc ctg aag tct ctg gaa gag aag 2928 Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys 965 970 975 gac cat atc cac cga gtc ctg gac aag atc aca gac act ttg atc cac 2976 Asp His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His 980 985 990 ctg atg gcc aag gca ggc ctg acc ctg cag cag cag cac cag cgg ctg 3024 Leu Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu 995 1000 1005 gcc cag ctc ctc ctc atc ctc tcc cac atc agg cac atg agt aac 3069 Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser Asn 1010 1015 1020 aaa ggc atg gag cat ctg tac agc atg aag tgc aag aac gtg gtg 3114 Lys Gly Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn Val Val 1025 1030 1035 ccc ctc tat gac ctg ctg ctg gag atg ctg gac gcc cac cgc cta 3159 Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala His Arg Leu 1040 1045 1050 cat gcg ccc act agc cgt gga ggg gca tcc gtg gag gag acg gac 3204 His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val Glu Glu Thr Asp 1055 1060 1065 caa agc cac ttg gcc act gcg ggc tct act tca tcg cat tcc ttg 3249 Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser His Ser Leu 1070 1075 1080 caa aag tat tac atc acg ggg gag gca gag ggt ttc cct gcc aca 3294 Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro Ala Thr 1085 1090 1095 gtc tga 3300 Val 4 1099 PRT Rat/human Chimera 4 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Gly Ala 225 230 235 240 Gly Ala Gly Ala Gly Ala Gly Ala Ile Ser Ala Leu Ile Leu Asp Ser 245 250 255 Lys Glu Ser Leu Ala Pro Pro Gly Arg Asp Glu Val Pro Gly Ser Leu 260 265 270 Leu Gly Gln Gly Arg Gly Ser Val Met Asp Phe Tyr Lys Ser Leu Arg 275 280 285 Gly Gly Ala Thr Val Lys Val Ser Ala Ser Ser Pro Ser Val Ala Ala 290 295 300 Ala Ser Gln Ala Asp Ser Lys Gln Gln Arg Ile Leu Leu Asp Phe Ser 305 310 315 320 Lys Gly Ser Thr Ser Asn Val Gln Gln Arg Gln Gln Gln Gln Gln Gln 325 330 335 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Gly Leu 340 345 350 Ser Lys Ala Val Ser Leu Ser Met Gly Leu Tyr Met Gly Glu Thr Glu 355 360 365 Thr Lys Val Met Gly Asn Asp Leu Gly Tyr Pro Gln Gln Gly Gln Leu 370 375 380 Gly Leu Ser Ser Gly Glu Thr Asp Phe Arg Leu Leu Glu Glu Ser Ile 385 390 395 400 Ala Asn Leu Asn Arg Ser Thr Ser Val Pro Glu Asn Pro Lys Ser Ser 405 410 415 Thr Ser Ala Thr Gly Cys Ala Thr Pro Thr Glu Lys Glu Phe Pro Lys 420 425 430 Thr His Ser Asp Ala Ser Ser Glu Gln Gln Asn Arg Lys Ser Gln Thr 435 440 445 Gly Thr Asn Gly Gly Ser Val Lys Leu Tyr Pro Thr Asp Gln Ser Thr 450 455 460 Phe Asp Leu Leu Lys Asp Leu Glu Phe Ser Ala Gly Ser Pro Ser Lys 465 470 475 480 Asp Thr Asn Glu Ser Pro Trp Arg Ser Asp Leu Leu Ile Asp Glu Asn 485 490 495 Leu Leu Ser Pro Leu Ala Gly Glu Asp Asp Pro Phe Leu Leu Glu Gly 500 505 510 Asn Thr Asn Glu Asp Cys Lys Pro Leu Ile Leu Pro Asp Thr Lys Pro 515 520 525 Lys Ile Lys Asp Thr Gly Asp Thr Ile Leu Ser Ser Pro Ser Ser Val 530 535 540 Ala Leu Pro Gln Val Lys Thr Glu Lys Asp Asp Phe Ile Glu Leu Cys 545 550 555 560 Thr Pro Gly Val Ile Lys Gln Glu Lys Leu Gly Pro Val Tyr Cys Gln 565 570 575 Ala Ser Phe Ser Gly Thr Asn Ile Ile Gly Asn Lys Met Ser Ala Ile 580 585 590 Ser Val His Gly Val Ser Thr Ser Gly Gly Gln Met Tyr His Tyr Asp 595 600 605 Met Asn Thr Ala Ser Leu Ser Gln Gln Gln Asp Gln Lys Pro Val Phe 610 615 620 Asn Val Ile Pro Pro Ile Pro Val Gly Ser Glu Asn Trp Asn Arg Cys 625 630 635 640 Gln Gly Ser Gly Glu Asp Ser Leu Thr Ser Leu Gly Ala Leu Asn Phe 645 650 655 Pro Gly Arg Ser Val Phe Ser Asn Gly Tyr Ser Ser Pro Gly Met Arg 660 665 670 Pro Asp Val Ser Ser Pro Pro Ser Ser Ser Ser Ala Ala Thr Gly Pro 675 680 685 Pro Pro Lys Leu Cys Leu Val Cys Ser Asp Glu Ala Ser Gly Cys His 690 695 700 Tyr Gly Val Leu Thr Cys Gly Ser Cys Lys Val Phe Phe Lys Arg Ala 705 710 715 720 Val Glu Gly Gln His Asn Tyr Leu Cys Ala Gly Arg Asn Asp Cys Ile 725 730 735 Ile Asp Lys Ile Arg Arg Lys Asn Cys Pro Ala Cys Arg Tyr Arg Lys 740 745 750 Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys Lys Lys 755 760 765 Ile Lys Gly Ile Gln Gln Ala Thr Ala Gly Val Ser Gln Asp Thr Ser 770 775 780 Glu Asn Pro Asn Lys Thr Ile Val Pro Ala Ala Leu Pro Gln Leu Thr 785 790 795 800 Pro Thr Leu Val Ser Leu Leu Glu Val Ile Glu Pro Glu Val Leu Tyr 805 810 815 Ala Gly Tyr Asp Ser Ser Val Ser Arg Gly Glu Pro Pro Ile Leu Tyr 820 825 830 Ser Glu Tyr Asp Pro Thr Arg Pro Phe Ser Glu Ala Ser Met Met Gly 835 840 845 Leu Leu Thr Asn Leu Ala Asp Arg Glu Leu Val His Met Ile Asn Trp 850 855 860 Ala Lys Arg Val Pro Gly Phe Val Asp Leu Thr Leu His Asp Gln Val 865 870 875 880 His Leu Leu Glu Cys Ala Trp Leu Glu Ile Leu Met Ile Gly Leu Val 885 890 895 Trp Arg Ser Met Glu His Pro Val Lys Leu Leu Phe Ala Pro Asn Leu 900 905 910 Leu Leu Asp Arg Asn Gln Gly Lys Cys Val Glu Gly Met Val Glu Ile 915 920 925 Phe Asp Met Leu Leu Ala Thr Ser Ser Arg Phe Arg Met Met Asn Leu 930 935 940 Gln Gly Glu Glu Phe Val Cys Leu Lys Ser Ile Ile Leu Leu Asn Ser 945 950 955 960 Gly Val Tyr Thr Phe Leu Ser Ser Thr Leu Lys Ser Leu Glu Glu Lys 965 970 975 Asp His Ile His Arg Val Leu Asp Lys Ile Thr Asp Thr Leu Ile His 980 985 990 Leu Met Ala Lys Ala Gly Leu Thr Leu Gln Gln Gln His Gln Arg Leu 995 1000 1005 Ala Gln Leu Leu Leu Ile Leu Ser His Ile Arg His Met Ser Asn 1010 1015 1020 Lys Gly Met Glu His Leu Tyr Ser Met Lys Cys Lys Asn Val Val 1025 1030 1035 Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala His Arg Leu 1040 1045 1050 His Ala Pro Thr Ser Arg Gly Gly Ala Ser Val Glu Glu Thr Asp 1055 1060 1065 Gln Ser His Leu Ala Thr Ala Gly Ser Thr Ser Ser His Ser Leu 1070 1075 1080 Gln Lys Tyr Tyr Ile Thr Gly Glu Ala Glu Gly Phe Pro Ala Thr 1085 1090 1095 Val 5 3273 DNA Rat/human Chimera CDS (1)..(3273) 5 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ctg acc tac ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag 240 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag aac 480 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggc agc 528 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag tcc 720 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 ggc gcg ctg atc ctg gac tcc aaa gaa tcc tta gct ccc cct ggt aga 768 Gly Ala Leu Ile Leu Asp Ser Lys Glu Ser Leu Ala Pro Pro Gly Arg 245 250 255 gac gaa gtc cct ggc agt ttg ctt ggc cag ggg agg ggg agc gta atg 816 Asp Glu Val Pro Gly Ser Leu Leu Gly Gln Gly Arg Gly Ser Val Met 260 265 270 gac ttt tat aaa agc ctg agg gga gga gct aca gtc aag gtt tct gca 864 Asp Phe Tyr Lys Ser Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala 275 280 285 tct tcg ccc tca gtg gct gct gct tct cag gca gat tcc aag cag cag 912 Ser Ser Pro Ser Val Ala Ala Ala Ser Gln Ala Asp Ser Lys Gln Gln 290 295 300 agg att ctc ctt gat ttc tcg aaa ggc tcc aca agc aat gtg cag cag 960 Arg Ile Leu Leu Asp Phe Ser Lys Gly Ser Thr Ser Asn Val Gln Gln 305 310 315 320 cga cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag 1008 Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 325 330 335 cag cag cag cag cca ggc tta tcc aaa gcc gtt tca ctg tcc atg ggg 1056 Gln Gln Gln Gln Pro Gly Leu Ser Lys Ala Val Ser Leu Ser Met Gly 340 345 350 ctg tat atg gga gag aca gaa aca aaa gtg atg ggg aat gac ttg ggc 1104 Leu Tyr Met Gly Glu Thr Glu Thr Lys Val Met Gly Asn Asp Leu Gly 355 360 365 tac cca cag cag ggc caa ctt ggc ctt tcc tct ggg gaa aca gac ttt 1152 Tyr Pro Gln Gln Gly Gln Leu Gly Leu Ser Ser Gly Glu Thr Asp Phe 370 375 380 cgg ctt ctg gaa gaa agc att gca aac ctc aat agg tcg acc agc gtt 1200 Arg Leu Leu Glu Glu Ser Ile Ala Asn Leu Asn Arg Ser Thr Ser Val 385 390 395 400 cca gag aac ccc aag agt tca acg tct gca act ggg tgt gct acc ccg 1248 Pro Glu Asn Pro Lys Ser Ser Thr Ser Ala Thr Gly Cys Ala Thr Pro 405 410 415 aca gag aag gag ttt ccc aaa act cac tcg gat gca tct tca gaa cag 1296 Thr Glu Lys Glu Phe Pro Lys Thr His Ser Asp Ala Ser Ser Glu Gln 420 425 430 caa aat cga aaa agc cag acc ggc acc aac gga ggc agt gtg aaa ttg 1344 Gln Asn Arg Lys Ser Gln Thr Gly Thr Asn Gly Gly Ser Val Lys Leu 435 440 445 tat ccc aca gac caa agc acc ttt gac ctc ttg aag gat ttg gag ttt 1392 Tyr Pro Thr Asp Gln Ser Thr Phe Asp Leu Leu Lys Asp Leu Glu Phe 450 455 460 tcc gct ggg tcc cca agt aaa gac aca aac gag agt ccc tgg aga tca 1440 Ser Ala Gly Ser Pro Ser Lys Asp Thr Asn Glu Ser Pro Trp Arg Ser 465 470 475 480 gat ctg ttg ata gat gaa aac ttg ctt tct cct ttg gcg gga gaa gat 1488 Asp Leu Leu Ile Asp Glu Asn Leu Leu Ser Pro Leu Ala Gly Glu Asp 485 490 495 gat cca ttc ctt ctc gaa ggg aac acg aat gag gat tgt aag cct ctt 1536 Asp Pro Phe Leu Leu Glu Gly Asn Thr Asn Glu Asp Cys Lys Pro Leu 500 505 510 att tta ccg gac act aaa cct aaa att aag gat act gga gat aca atc 1584 Ile Leu Pro Asp Thr Lys Pro Lys Ile Lys Asp Thr Gly Asp Thr Ile 515 520 525 tta tca agt ccc agc agt gtg gca cta ccc caa gtg aaa aca gaa aaa 1632 Leu Ser Ser Pro Ser Ser Val Ala Leu Pro Gln Val Lys Thr Glu Lys 530 535 540 gat gat ttc att gaa ctt tgc acc ccc ggg gta att aag caa gag aaa 1680 Asp Asp Phe Ile Glu Leu Cys Thr Pro Gly Val Ile Lys Gln Glu Lys 545 550 555 560 ctg ggc cca gtt tat tgt cag gca agc ttt tct ggg aca aat ata att 1728 Leu Gly Pro Val Tyr Cys Gln Ala Ser Phe Ser Gly Thr Asn Ile Ile 565 570 575 ggt aat aaa atg tct gcc att tct gtt cat ggt gtg agt acc tct gga 1776 Gly Asn Lys Met Ser Ala Ile Ser Val His Gly Val Ser Thr Ser Gly 580 585 590 gga cag atg tac cac tat gac atg aat aca gca tcc ctt tct cag cag 1824 Gly Gln Met Tyr His Tyr Asp Met Asn Thr Ala Ser Leu Ser Gln Gln 595 600 605 cag gat cag aag cct gtt ttt aat gtc att cca cca att cct gtt ggt 1872 Gln Asp Gln Lys Pro Val Phe Asn Val Ile Pro Pro Ile Pro Val Gly 610 615 620 tct gaa aac tgg aat agg tgc caa ggc tcc gga gag gac agc ctg act 1920 Ser Glu Asn Trp Asn Arg Cys Gln Gly Ser Gly Glu Asp Ser Leu Thr 625 630 635 640 tcc ttg ggg gct ctg aac ttc cca ggc cgg tca gtg ttt tct aat ggg 1968 Ser Leu Gly Ala Leu Asn Phe Pro Gly Arg Ser Val Phe Ser Asn Gly 645 650 655 tac tca agc cct gga atg aga cca gat gta agc tct cct cca tcc agc 2016 Tyr Ser Ser Pro Gly Met Arg Pro Asp Val Ser Ser Pro Pro Ser Ser 660 665 670 tcg tca gca gcc acg gga cca cct ccc aag ctc tgc ctg gtg tgc tcc 2064 Ser Ser Ala Ala Thr Gly Pro Pro Pro Lys Leu Cys Leu Val Cys Ser 675 680 685 gat gaa gct tca gga tgt cat tac ggg gtg ctg aca tgt gga agc tgc 2112 Asp Glu Ala Ser Gly Cys His Tyr Gly Val Leu Thr Cys Gly Ser Cys 690 695 700 aaa gta ttc ttt aaa aga gca gtg gaa gga cag cac aat tac ctt tgt 2160 Lys Val Phe Phe Lys Arg Ala Val Glu Gly Gln His Asn Tyr Leu Cys 705 710 715 720 gct gga aga aac gat tgc atc att gat aaa att cga agg aaa aac tgc 2208 Ala Gly Arg Asn Asp Cys Ile Ile Asp Lys Ile Arg Arg Lys Asn Cys 725 730 735 cca gca tgc cgc tat cgg aaa tgt ctt cag gct gga atg aac ctt gaa 2256 Pro Ala Cys Arg Tyr Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu 740 745 750 gct cga aaa aca aag aaa aaa atc aaa ggg att cag caa gcc act gca 2304 Ala Arg Lys Thr Lys Lys Lys Ile Lys Gly Ile Gln Gln Ala Thr Ala 755 760 765 gga gtc tca caa gac act tcg gaa aat cct aac aaa aca ata gtt cct 2352 Gly Val Ser Gln Asp Thr Ser Glu Asn Pro Asn Lys Thr Ile Val Pro 770 775 780 gca gca tta cca cag ctc acc cct acc ttg gtg tca ctg ctg gag gtg 2400 Ala Ala Leu Pro Gln Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val 785 790 795 800 att gaa ccc gag gtg ttg tat gca gga tat gat agc tct gtt tcg cga 2448 Ile Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Ser Arg 805 810 815 ggc ctc tcg agc aaa gcg cac cag gaa acc ttc cct gcc ctc tgc cag 2496 Gly Leu Ser Ser Lys Ala His Gln Glu Thr Phe Pro Ala Leu Cys Gln 820 825 830 ctg ggc aaa tac act acg aac aac agc tca gaa caa cgt gtc tct ctg 2544 Leu Gly Lys Tyr Thr Thr Asn Asn Ser Ser Glu Gln Arg Val Ser Leu 835 840 845 gac att gac ctc tgg gac aag ttc agt gaa ctc tcc acc aag tgc atc 2592 Asp Ile Asp Leu Trp Asp Lys Phe Ser Glu Leu Ser Thr Lys Cys Ile 850 855 860 att aag act gtg gag ttc gcc aag cag ctg ccc ggc ttc acc acc ctc 2640 Ile Lys Thr Val Glu Phe Ala Lys Gln Leu Pro Gly Phe Thr Thr Leu 865 870 875 880 acc atc gcc gac cag atc acc ctc ctc aag gct gcc tgc ctg gac atc 2688 Thr Ile Ala Asp Gln Ile Thr Leu Leu Lys Ala Ala Cys Leu Asp Ile 885 890 895 ctg atc ctg cgg atc tgc acg cgg tac acg ccc gag cag gac acc atg 2736 Leu Ile Leu Arg Ile Cys Thr Arg Tyr Thr Pro Glu Gln Asp Thr Met 900 905 910 acc ttc tcg gac ggg ctg acc ctg aac cgg acc cag atg cac aac gct 2784 Thr Phe Ser Asp Gly Leu Thr Leu Asn Arg Thr Gln Met His Asn Ala 915 920 925 ggc ttc ggc ccc ctc acc gac ctg gtc ttt gcc ttc gcc aac cag ctg 2832 Gly Phe Gly Pro Leu Thr Asp Leu Val Phe Ala Phe Ala Asn Gln Leu 930 935 940 ctg ccc ctg gag atg gat gat gcg gag acg ggg ctg ctc agc gcc atc 2880 Leu Pro Leu Glu Met Asp Asp Ala Glu Thr Gly Leu Leu Ser Ala Ile 945 950 955 960 tgc ctc atc tgc gga gac cgc cag gac ctg gag cag ccg gac cgg gtg 2928 Cys Leu Ile Cys Gly Asp Arg Gln Asp Leu Glu Gln Pro Asp Arg Val 965 970 975 gac atg ctg cag gag ccg ctg ctg gag gcg cta aag gtc tac gtg cgg 2976 Asp Met Leu Gln Glu Pro Leu Leu Glu Ala Leu Lys Val Tyr Val Arg 980 985 990 aag cgg agg ccc agc cgc ccc cac atg ttc ccc aag atg cta atg aag 3024 Lys Arg Arg Pro Ser Arg Pro His Met Phe Pro Lys Met Leu Met Lys 995 1000 1005 att act gac ctg cga agc atc agc gcc aag ggg gct gag cgg gtg 3069 Ile Thr Asp Leu Arg Ser Ile Ser Ala Lys Gly Ala Glu Arg Val 1010 1015 1020 atc acg ctg aag atg gag atc ccg ggc tcc atg ccg cct ctc atc 3114 Ile Thr Leu Lys Met Glu Ile Pro Gly Ser Met Pro Pro Leu Ile 1025 1030 1035 cag gaa atg ttg gag aac tca gag ggc ctg gac act ctg agc gga 3159 Gln Glu Met Leu Glu Asn Ser Glu Gly Leu Asp Thr Leu Ser Gly 1040 1045 1050 cag ccg ggg ggt ggg ggg cgg gac ggg ggt ggc ctg gcc ccc ccg 3204 Gln Pro Gly Gly Gly Gly Arg Asp Gly Gly Gly Leu Ala Pro Pro 1055 1060 1065 cca ggc agc tgt agc ccc agc ctc agc ccc agc tcc aac aga agc 3249 Pro Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser Ser Asn Arg Ser 1070 1075 1080 agc ccg gcc acc cac tcc ccg tga 3273 Ser Pro Ala Thr His Ser Pro 1085 1090 6 1090 PRT Rat/human Chimera 6 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser 225 230 235 240 Gly Ala Leu Ile Leu Asp Ser Lys Glu Ser Leu Ala Pro Pro Gly Arg 245 250 255 Asp Glu Val Pro Gly Ser Leu Leu Gly Gln Gly Arg Gly Ser Val Met 260 265 270 Asp Phe Tyr Lys Ser Leu Arg Gly Gly Ala Thr Val Lys Val Ser Ala 275 280 285 Ser Ser Pro Ser Val Ala Ala Ala Ser Gln Ala Asp Ser Lys Gln Gln 290 295 300 Arg Ile Leu Leu Asp Phe Ser Lys Gly Ser Thr Ser Asn Val Gln Gln 305 310 315 320 Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 325 330 335 Gln Gln Gln Gln Pro Gly Leu Ser Lys Ala Val Ser Leu Ser Met Gly 340 345 350 Leu Tyr Met Gly Glu Thr Glu Thr Lys Val Met Gly Asn Asp Leu Gly 355 360 365 Tyr Pro Gln Gln Gly Gln Leu Gly Leu Ser Ser Gly Glu Thr Asp Phe 370 375 380 Arg Leu Leu Glu Glu Ser Ile Ala Asn Leu Asn Arg Ser Thr Ser Val 385 390 395 400 Pro Glu Asn Pro Lys Ser Ser Thr Ser Ala Thr Gly Cys Ala Thr Pro 405 410 415 Thr Glu Lys Glu Phe Pro Lys Thr His Ser Asp Ala Ser Ser Glu Gln 420 425 430 Gln Asn Arg Lys Ser Gln Thr Gly Thr Asn Gly Gly Ser Val Lys Leu 435 440 445 Tyr Pro Thr Asp Gln Ser Thr Phe Asp Leu Leu Lys Asp Leu Glu Phe 450 455 460 Ser Ala Gly Ser Pro Ser Lys Asp Thr Asn Glu Ser Pro Trp Arg Ser 465 470 475 480 Asp Leu Leu Ile Asp Glu Asn Leu Leu Ser Pro Leu Ala Gly Glu Asp 485 490 495 Asp Pro Phe Leu Leu Glu Gly Asn Thr Asn Glu Asp Cys Lys Pro Leu 500 505 510 Ile Leu Pro Asp Thr Lys Pro Lys Ile Lys Asp Thr Gly Asp Thr Ile 515 520 525 Leu Ser Ser Pro Ser Ser Val Ala Leu Pro Gln Val Lys Thr Glu Lys 530 535 540 Asp Asp Phe Ile Glu Leu Cys Thr Pro Gly Val Ile Lys Gln Glu Lys 545 550 555 560 Leu Gly Pro Val Tyr Cys Gln Ala Ser Phe Ser Gly Thr Asn Ile Ile 565 570 575 Gly Asn Lys Met Ser Ala Ile Ser Val His Gly Val Ser Thr Ser Gly 580 585 590 Gly Gln Met Tyr His Tyr Asp Met Asn Thr Ala Ser Leu Ser Gln Gln 595 600 605 Gln Asp Gln Lys Pro Val Phe Asn Val Ile Pro Pro Ile Pro Val Gly 610 615 620 Ser Glu Asn Trp Asn Arg Cys Gln Gly Ser Gly Glu Asp Ser Leu Thr 625 630 635 640 Ser Leu Gly Ala Leu Asn Phe Pro Gly Arg Ser Val Phe Ser Asn Gly 645 650 655 Tyr Ser Ser Pro Gly Met Arg Pro Asp Val Ser Ser Pro Pro Ser Ser 660 665 670 Ser Ser Ala Ala Thr Gly Pro Pro Pro Lys Leu Cys Leu Val Cys Ser 675 680 685 Asp Glu Ala Ser Gly Cys His Tyr Gly Val Leu Thr Cys Gly Ser Cys 690 695 700 Lys Val Phe Phe Lys Arg Ala Val Glu Gly Gln His Asn Tyr Leu Cys 705 710 715 720 Ala Gly Arg Asn Asp Cys Ile Ile Asp Lys Ile Arg Arg Lys Asn Cys 725 730 735 Pro Ala Cys Arg Tyr Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu 740 745 750 Ala Arg Lys Thr Lys Lys Lys Ile Lys Gly Ile Gln Gln Ala Thr Ala 755 760 765 Gly Val Ser Gln Asp Thr Ser Glu Asn Pro Asn Lys Thr Ile Val Pro 770 775 780 Ala Ala Leu Pro Gln Leu Thr Pro Thr Leu Val Ser Leu Leu Glu Val 785 790 795 800 Ile Glu Pro Glu Val Leu Tyr Ala Gly Tyr Asp Ser Ser Val Ser Arg 805 810 815 Gly Leu Ser Ser Lys Ala His Gln Glu Thr Phe Pro Ala Leu Cys Gln 820 825 830 Leu Gly Lys Tyr Thr Thr Asn Asn Ser Ser Glu Gln Arg Val Ser Leu 835 840 845 Asp Ile Asp Leu Trp Asp Lys Phe Ser Glu Leu Ser Thr Lys Cys Ile 850 855 860 Ile Lys Thr Val Glu Phe Ala Lys Gln Leu Pro Gly Phe Thr Thr Leu 865 870 875 880 Thr Ile Ala Asp Gln Ile Thr Leu Leu Lys Ala Ala Cys Leu Asp Ile 885 890 895 Leu Ile Leu Arg Ile Cys Thr Arg Tyr Thr Pro Glu Gln Asp Thr Met 900 905 910 Thr Phe Ser Asp Gly Leu Thr Leu Asn Arg Thr Gln Met His Asn Ala 915 920 925 Gly Phe Gly Pro Leu Thr Asp Leu Val Phe Ala Phe Ala Asn Gln Leu 930 935 940 Leu Pro Leu Glu Met Asp Asp Ala Glu Thr Gly Leu Leu Ser Ala Ile 945 950 955 960 Cys Leu Ile Cys Gly Asp Arg Gln Asp Leu Glu Gln Pro Asp Arg Val 965 970 975 Asp Met Leu Gln Glu Pro Leu Leu Glu Ala Leu Lys Val Tyr Val Arg 980 985 990 Lys Arg Arg Pro Ser Arg Pro His Met Phe Pro Lys Met Leu Met Lys 995 1000 1005 Ile Thr Asp Leu Arg Ser Ile Ser Ala Lys Gly Ala Glu Arg Val 1010 1015 1020 Ile Thr Leu Lys Met Glu Ile Pro Gly Ser Met Pro Pro Leu Ile 1025 1030 1035 Gln Glu Met Leu Glu Asn Ser Glu Gly Leu Asp Thr Leu Ser Gly 1040 1045 1050 Gln Pro Gly Gly Gly Gly Arg Asp Gly Gly Gly Leu Ala Pro Pro 1055 1060 1065 Pro Gly Ser Cys Ser Pro Ser Leu Ser Pro Ser Ser Asn Arg Ser 1070 1075 1080 Ser Pro Ala Thr His Ser Pro 1085 1090 

We claim:
 1. A method for making a recombinant nuclear translocation protein, comprising: covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the cytoplasmic/nuclear translocation domain of the glucocorticoid receptor protein, (ii) a superfamily receptor DNA sequence coding for the ligand binding domain of a superfamily receptor protein, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera, wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence; and expressing said DNA chimera in an expression system to prepare said protein.
 2. The method of claim 1, further comprising: covalently connecting said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor utilizing a DNA linker sequence.
 3. The method of claim 2, wherein said DNA linker sequence is a fragment of a polylinker sequence.
 4. The method of claim 1, wherein said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence.
 5. The method of claim 4, further comprising: covalently connecting said glucocorticoid receptor DNA sequence and said nucleic acid sequence for a marker protein utilizing a DNA linker sequence.
 6. The method of claim 1, wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence of said glucocorticoid receptor DNA sequence.
 7. The method of claim 1, wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence.
 8. The method of claim 1, wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including DNA bases corresponding to about amino acid residue 570of said glucocorticoid receptor protein.
 9. The method of claim 1, wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence.
 10. The method of claim 1, wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence, the complete helix 3 sequence, and at most a fragment of helix 1 of said superfamily receptor DNA sequence.
 11. A protein produced by the process of: covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the 5′ end of the DNA sequence of the glucocorticoid receptor protein, through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence, (ii) a superfamily receptor DNA sequence coding for the 3′ end of the DNA sequence of a superfamily receptor protein, through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera, wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence, wherein said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence, and wherein said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor are covalently connected by a DNA linker sequence; and expressing said DNA chimera in an expression system to prepare said protein.
 12. A nucleic acid chimera comprising: a nucleic acid sequence which codes for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein; and a nucleic acid sequence which codes for the ligand binding domain of a superfamily receptor protein.
 13. The nucleic acid chimera of claim 12, additionally comprising: a nucleic acid sequence for a marker protein domain.
 14. The nucleic acid chimera of claim 13, wherein said marker protein domain encodes a fluorescent protein.
 15. The nucleic acid chimera of claim 12, wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence of said glucocorticoid receptor DNA sequence.
 16. The nucleic acid chimera of claim 12, wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence.
 17. The nucleic acid chimera of claim 12, wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including DNA bases corresponding to about amino acid residue 570of said glucocorticoid receptor protein.
 18. The nucleic acid chimera of claim 12, wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence.
 19. The nucleic acid chimera of claim 12, wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence, the complete helix 3 sequence, and at most a fragment of helix 1 of said superfamily receptor DNA sequence.
 20. The nucleic acid chimera of claim 12, wherein said ligand binding domain is the ligand binding domain of estrogen receptor.
 21. The nucleic acid chimera of claim 12, which is SEQ. ID NO.
 1. 22. The nucleic acid chimera of claim 12, wherein said ligand binding domain is the ligand binding domain of retinoic acid receptor.
 23. The nucleic acid chimera of claim 12, which is SEQ. ID NO.
 2. 24. A chimeric protein comprising two elements: a glucocorticoid receptor 5′ end, encompassing the nuclear translocation domain and helix 1; and a superfamily receptor 3′ end, encompassing the ligand binding domain and helix
 3. 25. The chimeric protein of claim 24, further comprising a marker protein domain.
 26. The chimeric protein of claim 25, wherein said marker protein domain encodes a fluorescent protein.
 27. The chimeric protein of claim 24, wherein said ligand binding domain is the ligand binding domain of estrogen receptor.
 28. The chimeric protein of claim 24, which is SEQ. ID NO.
 3. 29. The chimeric protein of claim 24, wherein said ligand binding domain is the ligand binding domain of retinoic acid receptor.
 30. The chimeric protein of claim 24, which is SEQ. ID NO.
 4. 31. A method for detecting a ligand of a superfamily receptor protein, which comprises: producing a nucleic acid vector encoding a nucleic acid chimera comprising three elements: a 5′ end of a glucocorticoid receptor, encompassing the nuclear translocation domain and helix 1, a 3′ end of a superfamily receptor, encompassing the ligand binding domain and helix 3, and a nucleic acid sequence for a marker protein domain; transfecting a eukaryotic cell with said nucleic acid vector; isolating a clonal population of cells that express a chimeric protein translated from said nucleic acid vector; contacting said cells with a sample compound or composition; and detecting the presence of cytoplasmic/nuclear translocation in response to a ligand of said ligand binding domain.
 32. The method of claim 31, wherein said marker protein domain encodes a fluorescent protein.
 33. A method for determining the concentration of a ligand of a labeled chimeric superfamily receptor protein, which comprises: producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain; transfecting a eukaryotic cell with said nucleic acid vector; isolating a clonal population of transfected cells that express a chimeric protein translated from said nucleic acid vector; contacting said transfected cells with a sample; scanning one or more test cell(s) to obtain signal data from said labeled protein; converting said signal data to obtain the cellular location of said labeled protein in said test cell(s); and analyzing said data using an analysis system having an algorithm to calculate changes in the distribution of said labeled protein between the cell cytoplasm and the cell nucleus of said test cell(s), said analysis system having the capability of providing an accurate reading of the concentration of a ligand.
 34. The method of claim 33, wherein said marker protein domain encodes a fluorescent protein.
 35. A kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising: a cell-based system which expresses a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; and a detection system for the detection of the translocation of said marker protein.
 36. The kit of claim 35, additionally comprising: one or more compounds and/or compositions which stably associate with said chimeric protein in the absence of a ligand for the ligand binding domain of said chimeric protein, and which dissociates from said chimeric protein in the presence of a ligand for the ligand binding domain of said chimeric protein.
 37. A kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising: a quantity of a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; a cell-free membrane system which restricts translocation of the chimeric protein when no ligand is bound to the ligand binding domain of said chimeric protein, and which permits translocation of the chimeric protein when the ligand binding domain of said chimeric protein is bound to its ligand; and a detection system for the detection of the translocation of said marker protein.
 38. The kit of claim 37, additionally comprising: one or more compounds and/or compositions which stably associate with said chimeric protein in the absence of a ligand for the ligand binding domain of said chimeric protein, and which dissociates from said chimeric protein in the presence of a ligand for the ligand binding domain of said chimeric protein.
 39. A method for diagnosis of defects in the nuclear transportation process, which comprises: producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain; transfecting a set of suspected defective cells with said nucleic acid vector; isolating a clonal population of said cells that express a chimeric protein translated from said nucleic acid vector; contacting said cells with a ligand of said ligand binding domain; and detecting the presence or absence of cytoplasmic/nuclear translocation in response to said ligand.
 40. A method for treating defective translocation of a superfamily receptor protein from the cytoplasm to the nucleus of a cell, in an animal in need thereof, comprising: producing a nucleic acid vector which is capable of being transcribed, and which encodes a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein and a nucleic acid sequence coding for the ligand binding domain of said superfamily receptor protein; and transfecting a target cell in said animal with said nucleic acid vector.
 41. A pharmaceutical composition comprising: (i) a chimeric protein comprising two elements: a glucocorticoid receptor 5′ end, encompassing the nuclear translocation domain and helix 1; and a superfamily receptor 3′ end, encompassing the ligand binding domain and helix 3; and (ii) a pharmaceutically acceptable carrier. 